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 JoVE Biology

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

1

1Sigma Aldrich

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    ChineseVersion

    Summary

    Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

    Date Published: 9/17/2008, Issue 19; doi: 10.3791/899

    Cite this Article

    Cupp-Enyard, C. Sigma's Non-specific Protease Activity Assay - Casein as a Substrate. J. Vis. Exp. (19), e899, doi:10.3791/899 (2008).

    Abstract

    Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.

    To view this article in Chinese, click here

    Protocol

    Before beginning the assay, we need to make sure that the following reagents are correctly prepared:

    1. A 50 mM Potassium Phosphate Buffer, pH 7.5. Prepare using 11.4 mg/ml of potassium phospate dibasic, trihydrate in purified water and adjusting pH with 1M HCl. This solution is placed at 37°C prior to use.
    2. A 0.65% weight/volume casein solution, prepared by mixing 6.5 mg/ml of casein in the 50 mM potassium phosphate buffer. Gradually increased the solution temperature with gentle stirring to 80-85 °C for about 10 minutes until a homogenous dispersion is achieved. It is very important not to boil the solution. The pH is then adjusted if necessary with NaOH and HCl.
    3. A 110 mM Trichloroacetic acid solution, prepared by diluting a 6.1N stock 1:55 with purified water. Trichloroacetic acid is a strong acid and should be handled with care.
    4. 0.5 mM Folin & Ciolcaltea's, or Folin's Phenol Reagent, which is the solution that will react with tyrosine to generate a measurable color change that will be directly related to the activity of proteases. Folin's Phenol Reagent is an acid and should be handled with care.
    5. A 500 mM Sodium Carbonate solution, prepared using 53 mg/ml of anyhydrous sodium carbonate in purified water.
      An enzyme diluent solution, which consists of 10 mM Sodium Acetate Buffer with 5mM Calcium Acetate, pH 7.5, at 37°C. This solution is what we use to dissolve solid protease samples or dilute enzyme solutions.
    6. 1.1 mM L-tyrosine Standard stock solution Prepared using 0.2 mg/ml L-tyrosine in purified water and heated gently until the tyrosine dissolves. As with the casein, do not boil this solution. Allow the L-tyrosine standard to cool to room temperature. This solution will be diluted further to make our standard curve.
    7. Protease solution. Immediately before use, dissolve protease in enzyme diluent solution prepared in step 6.

    If necessary, use a solid protease sample of predetermined activity, which is dissolved using enzyme diluent to 0.1-0.2 units/ml. This solution serves as a positive control for the quality control assay and as validation for the calculations we will perform to determine enzyme activity.

    Setting up the Protease Assay and Standard Curves

    1. To begin this assay, find suitable vials that will hold about 15 mls. For each enzyme that will be tested, 4 vials are needed. One vial will be used as a blank, and three others will be used to assay activity of three dilutions of the protease. Three dilutions are useful when checking final calculations against each other.
      1. To each set of four vials, add 5mls of our 0.65% casein solution. Let them equilibrate in a water bath at 37°C for about 5 minutes.
      2. Add varying volumes of enzyme solution that will be tested to three of the test sample vials, but not the blank. Mix by swirling and incubate for 37°C for exactly ten minutes. The protease activity and consequential liberation of tyrosine during this incubation time is what will be measured and compared between test samples.
    2. After this 10 minute incubation, add the 5 mls of the TCA reagent to each tube to stop the reaction. Then, add an appropriate volume of enzyme solution to each tube, even the blank, so that the final volume of enzyme solution in each tube is 1 ml. This is done to account for the absorbance value of the enzyme itself and to ensure that the final volume in each tube is equal. Incubate the solutions at 37°C for 30 minutes.


      During this 30 minute incubation, you may want to set up your tyrosine standard dilutions. Use 6 dram vials (dram vials can be substituted with polypropylene tubes) that can easily hold 8 mls. To the six vials, add the 1.1 mM tyrosine standard stock solutions with the following volumes in mls: 0.05, 0.10, 0.20, 0.40, 0.50. Don't add any tyrosine standard to the blank. Lower standards may be needed for impure test samples that will yield little color change. Once the tyrosine standard solution has been added, add an appropriate volume of purified water to each of the standards to bring the volume to 2 mls.
    3. After the 30 minute incubation, filter each of the test solutions and the blank using a 0.45 um polyethersulfone syringe filter. Filtration is required to remove any insolubles from the samples.
      1. Add the filtration 2 mls of the test samples and blank filtrate to 4 dram vials that can hold at least 8 mls. The same type of vial in which the standards were prepared can be used.
      2. To all of the vials containing the standards and standard blank, add 5mls of sodium carbonate. For best results, add 1 ml of Folin's reagent immediately afterwards.
      3. Add sodium carbonate to regulate any pH drop created by the addition of the Folin's reagent.
      4. Add sodium carbonate to the test samples and test blank. These solutions become cloudy after the addition of sodium carbonate. Add the Folin's reagent, which will react primarily with free tyrosine.
      5. Mix the dram vials by swirling and incubate at 37°C for 30 minutes.
      After this incubation, you should notice that the standards have a gradation of color correlating with the amount of tyrosine added; the highest concentrations of tyrosine appearing darkest. You can also notice appreciable color change in our test samples. 2mls of these solutions are filtered using a 0.45 um polyethersulfone syringe filter into suitable cuvettes. Now that the assay is performed, you can proceed to the spectrophotometer to record our absorbance values.

    Measuring Absorbance and Calculating Enzyme Activity

    1. The absorbance of the samples is measured by a spectrophotometer using a wavelength of 660nm. The light path is set to 1cm. Record the absorbance values for the standards, standard blank, the different test samples, and test blank. Once all of the data has been collected, the standard curve can be created. In order to generate the curve, difference in absorbance between the standard and standard blank must be calculated. This is the absorbance value attributable to the amount of tyrosine in the standard solutions. After this simple calculation, create the standard curve using a graphing program to plot the change in absorbance of our standards on the Y axis, versus the amount in micromoles for each of our 5 standards on the X axis.

      Volume of Tyrosine Standard uMoles Tyrosine
      0.05 0.055
      0.10 0.111
      0.20 0.221
      0.40 0.442
      0.50 0.553
    2. After data points have been entered, generate a line of best fit and corresponding slope equation.


      Find the change in absorbance in the test samples by calculating the difference between the test sample absorbance and the absorbance of the test blank. Inserting the absorbance value for one of the test samples into the slope equation and solving will result in the micromoles of tyrosine liberated during this particular proteolytic reaction. To get the activity of enzyme in units per/ml, perform the following calculation:


      (umole tyrosine equivalents released) x (11)
      Units/ml Enzyme = __________________________________________

      (1) x (10) x (2)


      11= Total volume (in milliliters) of assay
      10= Time of assay (in minutes) as per the Unit definition
      1= Volume of Enzyme (in milliliters) of enzyme used
      2= Volume (in milliliters) used in Colorimetric Determination


      Take the number of micromoles tyrosine equivalents released obtained from the slope equation and multiply it by the total volume of the assay in mls, which in our case is 11mls. Divide this value by three other quantities: the time of the assay, which we ran for 10 minutes, the volume of enzyme used in the assay, which was varied (let's use 1ml), the volume of milliliters used in colorimetric detection, which may differ based on your cuvette We used 2 mls.
    3. Micromoles of tyrosine divided by time in minutes yields measurement of protease activity called "units". We can cancel out the units for volume measurement in the numerator and denominator, leaving a measurment of enzyme activity in terms of units/ml. In order to determine the activity in a solid protease sample diluted in enzyme diluent, we divide our activity in units/ml by the concentration of solid used in this assay originally in mg/ml., leaving us with activity in terms of units/mg.


      Units/ml enzyme
      Units/mg solid = _____________________

      mg solid/ ml enzyme

    Discussion

    We've just shown you how to analyze protease activity using Sigma's universal protease activity assay. In addition, this assay is useful to ensure that our proteases have precisely determined activity before you receive them for your experiments. As you have seen, when doing this procedure, it's of paramount importance to remember to heat both the casein and tyrosine solutions slowly and not to boil them. Also, it's critical to prepare different blanks for both your standards and for each test sample that you have.

    Disclosures

    Materials

    Name Company Catalog Number Comments
    Protease Sigma-Aldrich P4630
    Potassium Phosphate, Dibasic, Trihydrate Sigma-Aldrich P5504
    Casein Sigma-Aldrich C7078
    Trichloroacetic Acid Sigma-Aldrich T0699
    Folin & Ciocalteu’s Phenol Reagent Sigma-Aldrich F9252
    Sodium Carbonate, Anhydrous Sigma-Aldrich S2127
    Sodium Acetate, Trihydrate Sigma-Aldrich S8625
    Calcium Acetate Sigma-Aldrich C1000
    L-Tyrosine, Free Base Sigma-Aldrich T3754
    Protease Profiler Kit Sigma-Aldrich PP0500
    Protease Fluorescent Detection Kit Sigma-Aldrich PF0100

    References

    1. Anson, M.L., (1938) J. Gen. Physiol. 22, 79-89

    2. Folin, O. and Ciocalteau, V., (1929) J. Biol. Chem. 73, 627

    Comments

    135 Comments

    dear mam, This video was relly very useful for me to perform the assay... but i wonder what is the use of sodium carbonate here... can u pls reply me through mail. thank u.                                                                                                                                                     G.Hemamalini                                                                                                                                                     M.Sc biotechnology
    Reply

    Posted by: AnonymousJanuary 23, 2009, 4:49 AM

    good day! this is janice averilla.i just would like to ask if you have a list of amino acid components their corresponding percentage in casein. thanks.
    Reply

    Posted by: AnonymousMay 2, 2009, 5:00 AM

    Dear Janice, The amino acid components that make up the casein in this assay is not an important factor for completing this assay correctly.  If you are inquiring about a specific casein amino acid sequence of a particular species I would recommend querying a site such as www.uniprot.org for the protein sequence and species you require.                                                                                                        Sigma Aldrich
    Reply

    Posted by: Nathan A.May 4, 2009, 1:48 PM

    Dear G. Hemamalini, We are happy to hear that you found the video useful.  Sodium carbonate is used in this assay to provide the proper pH environment for color development using the Folin & Ciocaleu's reagent.                                                                                                     Sigma Aldrich
    Reply

    Posted by: Nathan A.May 4, 2009, 1:50 PM

    Sigma protease assay kit is based on casein as substrate and measure released tyrosin. I want to check a putative protease with unknown specificity. Please could you comment on the following questions. 1) Can I be sure that your kit will detect ANY protease? ²) What if casein dŒs not contain recongnition sites for a particular protease? 3) Same as above, but if activity requires particular conditions (pH, divalent cation(s). 4) Taking into account these limitations, why do you consider this as an adequate test for detection of general proteolytic activities? 5) Are there any other limitations of the assay? 6) Can you tell what proteases (classes, families etc) are actually detectable? Thanks
    Reply

    Posted by: AnonymousMay 6, 2009, 1:24 PM

    Casein has been used for decades in our labs for a diverse range of proteases. We have never seen a protease that casein was not cleaved by. However it is certainly possible that a very specific protease may not cleave caseins. This being the case, there is no known universal protease substrate for all concievable proteases. Casein is suitable for detection of serine, cysteine, metallo, and aspartic proteases; however, modifications may be required to detect some specific proteases.     Modifications to the procedure may include pH adjustments, the addition of metal ions, or a reformulation of the incubation buffer. The researcher must determine the optimal procedure conditions for the protease specific to their application. This procedure will detect 0.1 unit/ml (approximately ² mg) of trypsin with a 10 minute incubation time. This sensitivity may be increased by modifications, such as increased incubation time. If even higher sensitivity is required, the Protease Fluorescent Detection Kit (Product Code PF0100) is recommended.  
    Reply

    Posted by: Nathan A.May 7, 2009, 3:12 PM

    Hi,
    Would it be possible to use a larger subrtrate than Casein? I am using a broad specificity protease (papain) and need to use a large substrate (>~50kda).
    Thanks,
    Anthony
    Reply

    Posted by: AnonymousJune 30, 2009, 3:31 PM

    Hello Anthony,
    Using a larger substrate should be fine. You just want to ensure two things: that the protein has cleavage sites specific to papain but as you already noted papain has a broad specificity and that the protein can be precipitated by TCA.
    Reply

    Posted by: Nathan A.July 2, 2009, 9:47 AM

    dear mam,
    i have followed reported procedure for assay of serratiopeptidase which is little different than above mention procedure but unfortunately i am not getting peaks at 660 nm.important thing to notice is that i am not even getting bluish color at the end of assay that i can clearly see in the video. i tried to plot tyrosine reference curve but same problem persist.what may be the possible reasons behind this ?
    Ajinkya Kulkarni
    ajinkyakulkarni4u@gmail.com
    Reply

    Posted by: ajinkya k.November 25, 2009, 1:26 PM

    Dear Ajinkya

    Since you did not get color develoment with the protease sampls or the tyrosine standard curve, It appears the problem you are experiencing is due to color development using the Folin's Reagent step.

    A quick way to determine the source of the problem may be to repeat one tube of standard #4, and look for the blue color development.
    If no color is seen, then I would suggest the following procedure to find which reagent in this step is the cause of the problem:

    Confirm the concentration of the tyrosine standard (1.1 mM)
    Make new 500 mM sodium carbonate (sigma S5444) (do not use sodium bicarbonate)
    Check the appearance of the Folin's Reagent and make a fresh 1:3 dilution with water (The reagent should be bright
    yellow in color. Discard any reagent that has acquired a green tint)

    Add the reagents to a tube in the same order they are stated in the instructions:
    50ul tyrosine standard
    ²00 ul water
    6²5 ul sodium carbonate
    1²5ul of diluted folin's Reagent
    Reply

    Posted by: Nathan A.November 30, 2009, 4:26 PM

    dear Nathan,
    thank you very much. problem was with sodium carbonate. strength of sodium carbonate that i was using was wrong. i was following method given in indian pharmacopŒia which is quite same as above method is, in that they have suggested to use 0.6% w/v sodium carbonate which is actually 6% w/v.
    Reply

    Posted by: AnonymousJanuary 1, 2010, 12:20 PM


    ...............................................................................................................................................



















    in this assay, you are taking 1.1mM as the stock of tyrosine. but then how can you put micromoles in the graph when plotting for standard graph.

    Reply

    Posted by: AnonymousNovember 28, 2009, 6:52 AM

    The Tyrosine Standard curve is expressed in micromoles
    As an example calculation we can use Tyrosine Standard # 4

    50ul (volume added of the standard) X 1.1mM (which is the same as 0.0011 micromoles/ul) = 0.055 micromoles
    In the figure for the tyrosine standard curve, the last point (which corresponds to Standard #4) is at 0.055 micromoles
    Reply

    Posted by: Nathan A.November 30, 2009, 4:27 PM

    conversion of µg/ml to µmoles/ml of tyrosine
    1.concentration of tyrosine soln is 0.²mg/ml i.e. ²00µg/ml
    ².divide concentration by mol.wt of tyrosine (181.19) ²00/ 181.19 = 1.1µmoles/ml
    3 if we are taking 0.1 ml of tyrosine solution it contains 0.111µmoles/ml
    is my calculation is correct?
    Reply

    Posted by: AnonymousJanuary 20, 2010, 2:19 AM

    conversion of micrograms/ml to micromoles/ml of tyrosine
    1.concentration of tyrosine soln is 0.²mg/ml i.e. ²00 micrograms/ml
    ².divide concentration by mol.wt of tyrosine (181.19) ²00/ 181.19 = 1.1 micromoles/ml
    3 if we are taking 0.1 ml of tyrosine solution it contains 0.111micromoles/ml
    is my calculation is correct?
    microgram sign is not visible in previous reply so posted comment again.
    Reply

    Posted by: AnonymousJanuary 20, 2010, 2:23 AM

    Here is a math check
    I do everything in moles and moles per liter
    0.²mg/ml=0.²g/L
    (0.²g/L)/(181.19g/mole)=0.0011moles/L=1.1millimoles/L=1.1micromoles/ml

    Your math is correct in your steps 1 and ². ...For step 3 however, please note that 0.1ml of a 1.1micromole/ml solution contains a total of 0.11micromoles but the concentration is not 0.11 micromoles/ml unless the solution was further diluted 10X.
    Reply

    Posted by: Nathan A.January 20, 2010, 1:48 PM

    yes ,i understood.

    Posted by: AnonymousFebruary 4, 2010, 11:59 PM

    "². After this 10 minute incubation, add the 5 mls of the TCA reagent to each tube to stop the reaction. Then, add an appropriate volume of enzyme solution to each tube, even the blank, so that the final volume of enzyme solution in each tube is 1 ml. This is done to account for the absorbance value of the enzyme itself and to ensure that the final volume in each tube is equal. Incubate the solutions at 37°C for 30 minutes."
    Why should I add an appropriate volume of enzyme solution to each tube, even the blank ? Ok, you said :This is done to account for the absorbance value of the enzyme itself and to ensure that the final volume in each tube is equal. But I did not get the point. Would you please explain more? thanks.
    Reply

    Posted by: mohamed s.November 28, 2009, 2:22 PM

    The additional enzyme is added to the blank and test samples after the reaction is stopped with TCA to provide an equal amount of enzyme in each tube.
    This is important because:
    It is possible that your enzyme could have some endogenous absorbance at 660 nm
    It may be that your enzyme sample may have trace amounts of tyrosine than need to be blanked out equally in all test samples.
    Reply

    Posted by: Nathan A.November 30, 2009, 4:28 PM

    Can I use this procedure for estimation of protease enzyme of thermophilic organisms?pl reply soon.
    Reply

    Posted by: AnonymousDecember 6, 2009, 11:37 AM

    Yes, you can use this procedure for proteases from thermophillic organisms.
    Reply

    Posted by: Nathan A.December 9, 2009, 1:35 PM

    Can I use the same estimation procedure for thermophilic organism's protease enzyme?
    Reply

    Posted by: AnonymousDecember 6, 2009, 11:40 AM

    dear mam,
    can you pls reply me about the principle of each chemicals which is used in this protease assay procedure
    Reply

    Posted by: AnonymousJanuary 5, 2010, 8:20 PM

    dear mam,
    In my lab, I have folin ciocalteu's phenol reagent ( ² N) . So how can I make it 0.5 mM ? I have searched for it's m.w. on the net but coulld not find it. So looking forward for your answer mam. Plz reply soon.
    Reply

    Posted by: AnonymousJanuary 10, 2010, 6:27 AM

    Folin ciocalteu's phenol reagent is a mixture of compounds, the molarity refers to the concentration of acid in the mixture. Simply dilute one part of the reagent with 3 parts deionized water.
    Reply

    Posted by: Nathan A.January 11, 2010, 12:01 PM

    Hello
    I use Folin phenol & Ciocalteu&#x²019;s reagen that is made by Merk, but its normality is not notified.
    How can I know that ? I know its density but I can't find the molecular weight anywhere.
    would you please tell me its molecular weight. thank you.
    Reply

    Posted by: AnonymousJanuary 24, 2010, 12:44 PM

    Hello
    I use Folin phenol & Ciocalteu&#x²019;s reagen that is made by Merk, but its normality is not notified.
    How can I know that ? I know its density but I can't find the molecular weight anywhere.
    would you please tell me its molecular weight. thank you.
    Reply

    Posted by: amin a.January 24, 2010, 12:57 PM

    Hello
    I use Folin phenol & Ciocalteu&#x²019;s reagen that is made by Merk, but its normality is not notified.
    How can I know that ? I know its density but I can't find the molecular weight anywhere.
    would you please tell me its molecular weight. thank you.
    Reply

    Posted by: amin a.January 24, 2010, 12:59 PM

    dear mam,
    This protocol was very useful for me to do my project, i want to know the use of TCA in this assay.
    D.revathi
    B.tech,biotechnology
    Reply

    Posted by: AnonymousFebruary 3, 2010, 4:08 AM

    ma'am, I have ploted a graph of O.D.( y axis) vs tyrosin concentration in microgram/ml(x axis).now I have converted microgram/ml tyrosin content into micromol/ml as below.
    if ²00 microgram/ml = 1.1 mM , then 135 microgram/ml ( the concentration from standard currve as a result) =0.74²5 mM
    now 0.74²5 mM is multiplied to 1000 to convert it into 74².5 micromol.
    Is it right or not .pl reply soon.
    Reply

    Posted by: AnonymousFebruary 21, 2010, 12:59 PM

    135ug/ml is equivalent to a 74² micromolar concentration of tyrosine.
    Reply

    Posted by: Nathan A.February 22, 2010, 10:48 AM

    How can i solve, esstimation of protease activity at optimum ph?
    Reply

    Posted by: AnonymousFebruary 25, 2010, 12:24 PM

    Hi, I really like the video. But I need to check the activity of the enzyme in a detergent. How I can do it , do I have to make the solution sample as a detergent? Could you please help me.

    Carotaba84@yahoo.com.mx
    Reply

    Posted by: AnonymousMay 5, 2010, 11:01 AM

    The detergent should be included in the initial reaction mixture to yield the final concentration at which the protease activity is to be tested.

    I would suggest adding the detergent to the Casein protease detection substrate solution initially (since it is the largest volume added to the reaction mix).
    I would suggest including the following additional "reaction tubes".
    Two blanks with and without detergent (this will indicate if any changes in the final absorption readings are affected by the detergent)
    Two sets of samples and controls with and without detergent. (use a protease that is known to be active in your detergent as the control)...(This will tell you the difference in activity with and without detergent)

    After the initial protease digestion reactions all steps and reagents (starting with the addition of the TCA) can remain the same as described in the video
    Reply

    Posted by: Nathan A.May 5, 2010, 1:34 PM

    Hi,

    I need someone to help me , but I do not know how I should obtain the equation of the standar curve using linear regression. Could someone expplain me because I am really confuse and I need to determinate the activity of enzymes.

    Thanks a lot.
    Reply

    Posted by: AnonymousMay 12, 2010, 4:05 PM

    Hi,

    I need someone to help me , but I do not know how I should obtain the equation of the standar curve using linear regression. Could someone expplain me because I am really confuse and I need to determinate the activity of enzymes.

    Thanks a lot.
    Reply

    Posted by: AnonymousMay 13, 2010, 10:58 AM

    I have an crude protease extract, however, I don't know which classes of protease dŒs it contain. Maybe all of them or one of them. If I use this procedure, do I find total protease activity? Or do I need to modify the procedure for each of the protease class (serine, cysteine, metallo, and aspartic proteases) to guess total activity? So, four different analysis? What do you suggest?
    Reply

    Posted by: AnonymousJune 5, 2010, 10:43 AM

    You should find total protease activity using this procedure and will not need to modify for each class of protease. Casein, the substrate used in this procedure has been used for decades in our labs for a diverse range of proteases. We have never seen a protease that casein was not cleaved by. However it is certainly possible that a very specific protease may not cleave casein. Although this would be very rare.
    Reply

    Posted by: AnonymousJune 7, 2010, 11:46 AM

    Thank you very much for the explanation. But I have also one question. The pH choosen for the test is 7.5. If there are acid and alkaline proteases in my enzyme extract, will I still determine the total protease activity accurately?
    Reply

    Posted by: AnonymousJune 7, 2010, 3:25 PM

    Dear madam,
    i am having one doubt. I am doing my research on the production of alkaline protease. Now i am screening various carbon and nitrogen sources to get maximum yield. Can i use this procedure for enzyme assay? Because the carbon and nitrogen sources i am varying for my studies. The nature of enzyme may be vary. Whether this procedure is adopted for my studies or not? If not tell me the procedure for assay.
    Reply

    Posted by: AnonymousJune 10, 2010, 3:06 AM

    hello
    I am doing research on microbial alkaline protease production. i have seen your video. it is good and informative. Whether this procedure can be used for all kind of alkaline protease or not? Suppose i am varying the carbon and nitrogen sources to get maximum yield of alkaline protease, to analyze the activity, whether this procedure can be used or not?
    Reply

    Posted by: Ganesh Moorthy I.June 10, 2010, 5:02 AM

    This procedure will in fact work with all kinds of alkaline proteases. Varying the carbon or nitrogen source should not impact the effectiveness of this procedure.
    Reply

    Posted by: Nathan A.June 10, 2010, 3:57 PM

    hello
    I new in this area. so I hope u can help me bcz previously i took polymer and physical chemistry.
    I hv ² question.. can I know how to prepare protease solution. 1) crude extrct (dnt hv specific activity)
    ²) from cysteine (sample from sigma). because dont hv unit/mg
    If is true if I weight the cysteine for 1 g and dilute with buffer untill 100ml. 1: 100.

    PhD in bio-catalyst
    Reply

    Posted by: AnonymousJuly 9, 2010, 7:50 AM

    Hello,
    What is the source of your extract containing protease, and how was it prepared? For unkonwn sources of protease activity, several dilutions must be measured and may lead to subsequent refinement of the amount and dilution of the protease.

    I do not understand the significance of cysteine in your procedure?
    Reply

    Posted by: Nathan A.July 12, 2010, 3:10 PM

    good morning Nathan.

    My source for extract is mango. I prepared it by extracted it in pH 7.5, 5 mM calcium chloride and 4°C. after that I used centrifuge, ammonium sulphate, dialysis and anion exchanger chromatography. The solution for dilution is buffer solution or water. If we used water, the enzyme denatured or not if the pH of the enzyme pH9.0.

    for cysteine, I took it as a protease to replace the subtilisn. this is because in my lab, only have it. As your all know, the protease have 5 types. so the protease activity inside the video are suitable for all the types.

    Thank you so much
    Reply

    Posted by: AnonymousJuly 22, 2010, 7:15 PM

    Hello Jimy,
    This helps me understand a bit more but there are still some things I'm a little confused about. Could you contact me directly at nathan.allen@sial.com? That way we can work with one of Sigma's enzyme specialists to find a solution for your experiment. Thanks.
    Reply

    Posted by: Nathan A.July 23, 2010, 10:06 AM

    i really need to know,can i use this activity assay procedure for Sigma product P6110 (Protease from Aspergillus oryzae)?i did try to find the activity assay procedure specifically for this P6110 product but didnt find any.please reply me very soon.thank you very much
    Reply

    Posted by: AnonymousJuly 25, 2010, 10:29 AM

    please give me information about the enzymes that specifically or non-specifically digest the casein to casomorphin -7
    Reply

    Posted by: AnonymousAugust 6, 2010, 5:42 AM

    I have a question, DŒs The tryptophan bind to Folin reagen and absorb to 660 nm?
    Thanks...
    Reply

    Posted by: Olga P.October 13, 2010, 2:06 AM

    Yes, that is correct.
    Reply

    Posted by: AnonymousOctober 18, 2010, 12:55 PM

    Dear mam
    do you suggest us few more protease assays which are less time consuming and cheaper to perform for my research
    may i know the importance of each and every ingredient used in this assay?
    thanks and regards
    swapna
    M.Tech (Bio-Tech)
    Reply

    Posted by: AnonymousDecember 7, 2010, 12:33 AM

    pls give example with complete calcultion.
    Regards
    Anil
    Reply

    Posted by: AnonymousDecember 13, 2010, 12:15 AM

    I am doing the same work, I some where referred that O.D. for tyrosine standard should be taken at 540nm as the concentration is
    Reply

    Posted by: AnonymousApril 29, 2011, 6:57 AM

    Dear sir/Madam
    I have a question.Due to indefinite molecular weight of casein, i found difficulty in calculating km and vmax for protease. Can you please help me to complete my calculation?
    1.As per your protocol 6.5 mg/ml of casein were prepared.When i performed the different substrate concentration on protease i found 14.5mg as km.In other protocols (substrate different), they were showed the [S] concentration in micromoles. Hence i couldnot give supportive studies for my work.what to do. please help.
    Reply

    Posted by: AnonymousMay 18, 2011, 11:26 AM

    The Casein which is used as a substrate in this assay consists of a mixture of four different isoforms with molecular weights of 19, ²², ²4, and ²5 kDa. If you used the average molecular weight of ²²,500 daltons, a concentration of 14.5 mg/ml would be equivalent to 0.64 mM.
    Reply

    Posted by: Nathan A.May 18, 2011, 1:20 PM

    Dear Nathan
    Thank you very much. How did you convert 14.5 mg/ml to 0.64 mM ?.Please reply.Because i have many calculations similar to this.

    Reply

    Posted by: AnonymousMay 18, 2011, 11:51 PM

    Hello,
    The Casein which is used as a substrate in this assay consists of a mixture of four different isoforms with molecular weights of 19, ²², ²4, and ²5 kDa. If you used the average molecular weight of ²²,500 daltons, a concentration of 14.5 mg/ml would be equivalent to 0.64 mM. This would be calculated as:
    14.5 mg/ml = 14.5 g/L Divide this value by the MW of ²²,500 (14.5/²²,500) = 6.4 x 10(-4)M or 0.64 mM.
    Reply

    Posted by: Nathan A.May 19, 2011, 10:13 AM

    thank you sir
    Reply

    Posted by: AnonymousMay 19, 2011, 12:48 PM

    Dear Sir
    Your protocol is very usefull for me. I have one doubt.In my experiment, i took pond water as a test solution for protease assay. in this i got good colouration of test solution, when compare to standards. What it means ? ( Is that represents that much of proteases in pond water?)
    Reply

    Posted by: AnonymousJuly 6, 2011, 10:48 AM

    Dear Sir
    Your protocol is very usefull for me. I have one doubt.In my experiment, i took pond water as a test solution for protease assay. in this i got good colouration of test solution, when compare to standards. What it means ? ( Is that represents that much of proteases in pond water?)

    Reply

    Posted by: AnonymousJuly 10, 2011, 1:52 PM

    Hello,
    You may want to run a sample control (1 ml sample solution plus 5 ml of substrate blank-50 mM potassium phosphate buffer only), just to insure that the sample (pond water) is not contributing to the absorbance at 660 nm. If this absorbance is baseline, then any color at 660 nm can be attributed to protease present in the sample.
    Reply

    Posted by: Nathan A.July 11, 2011, 1:46 PM

    Dear sir
    Please explain in detail. I couldn't follow.
    Reply

    Posted by: AnonymousJuly 13, 2011, 10:51 AM

    hello
    I am doing research on some protease activity. it is very good and informative.but I have one question. Whether this total volume can be changed or not? I want to make a total volume about 100 or ²00 ul.
    Can I change the total volume? and also get same result?
    Reply

    Posted by: AnonymousAugust 12, 2011, 2:39 AM

    Hello Lauren,
    Unfortunately, Sigma has never tried this protease activity assay at this smaller scale. Theoretically it would be possible, but it might be rather difficult to filter the small volumes. However, when scaling down (volumes) an assay, there can be other concerns such as protein out-gassing or insufficient substrate mixing. It dŒs not always work. I wish I had a definitive answer for you but all I can say is to give it a try.
    Reply

    Posted by: Nathan A.August 15, 2011, 10:21 AM

    Can I dilute protease solid by another diluents insteads by using Sodium Acetate and Calcium Acetate?
    If can, what can diluents use?
    Thanks in advanced for your reply!
    Reply

    Posted by: thu t.September 6, 2011, 2:47 AM

    Hello,
    This assay has been optimized for measuring the activity of neutral proteases offered by Sigma, which is why the recommended reconstitution buffer for the enzyme is 10 mM Sodium Acetate Buffer with 5 mM Calcium Acetate, pH 7.5. Calcium is a required cofactor for these proteases also. The assay buffer consists of 50 mM Potassium Phosphate buffer, pH 7.5. I would not recommend replacing the sodium acetate with the potassium phosphate, as the calcium may precipitate. The potassium phosphate is required in the assay because it is needed to solubilize the Casein. I would not recommend changing buffers. However, if there is a specific reason for not using the sodium acetate buffer for reconstituting the protease, you could try using 10 mM Tris-HCl, pH 7.5.
    Reply

    Posted by: Nathan A.September 6, 2011, 3:20 PM

    Dear Nathan Allen,
    Thank you very much for your reply!
    Reply

    Posted by: thu t.September 7, 2011, 8:23 AM

    I have protease enzyme that has 3.6 units/mg of protease enzyme solid
    so, if I want diluting enzyme that has 0.1 unit/ml
    so that I calculate 0.1 / 3.6 = 0.0²778 mg/ml
    and 0.00²778 g/100ml , is that right or wrong?
    Thanks in advanced for your reply !
    Thu
    Reply

    Posted by: thu t.September 12, 2011, 3:59 AM

    I have protease enzyme that has 3.6 units/mg of protease enzyme solid
    so, if I want diluting enzyme that has 0.1 unit/ml
    so that I calculate 0.1 / 3.6 = 0.0²778 mg/ml
    and 0.00²778 g/100ml , is that right or wrong?
    Thanks in advanced for your reply !
    Thu
    Reply

    Posted by: thu t.September 12, 2011, 3:26 AM

    If the activity of the protease is 3.6 units/mg solid, I would recommend initially reconstituting it at 1 mg/ml, which would be equivalent to 3.6 units/ml. Next dilute it to a final volume of 36 ml. The resulting concentration will be 0.1unit/ml. This concentration would also be equal to 0.0²78 mg/ml. I would not recommend performing a weigh-up of less than 1 mg of the enzyme
    Reply

    Posted by: Nathan A.September 13, 2011, 3:26 PM

    Dear Nathan Allen,
    Thank you very much for your reply!
    Reply

    Posted by: thu t.September 14, 2011, 7:52 AM

    yes, it is right.
    Reply

    Posted by: AnonymousFebruary 29, 2012, 3:04 AM

    Can I use this method for Protease Activity Assay?
    The sample, 0.05 ml of Flavourzyme, and the standard was separated into the mixture containing 5 ml of 0.1 M Tris ²11; HCl buffer pH 8.5 and 1.0 ml of 5% casein. After 30 minutes of incubation at 30C, the reaction mixture was terminated by addition of 0.5 ml of 10% TCA and kept in an ice bath for 10 minutes. The mixtures were the filtered through Whatman No. 50 filter paper. Filter (²ml), 0.² M Alkaline sodium carbonate (5ml) and 1 ml of Flolin and ciocalteu²17;s phenol reagent (²5ml original solution mixed with 50 ml de ²11; ionized water) were mixed and incubated at 37C for 30 minutes. The standard curve was then constructed.
    Can I also use 0.1M Tris - HCl, pH 8.5 to dilute solid protease and add 36ml of 0.1M Tris - HCl into 1mg of solid protease to get 0.1 unit/ml?
    Can I keep mixture in refrigerator insteads ice baht for 10 minutes?
    would you please tell me this method can use or not? thank you very much.
    Mary,
    Reply

    Posted by: AnonymousSeptember 20, 2011, 4:35 AM

    would you please tell me this method can use or not? thank you very much.
    Mary,
    Reply

    Posted by: AnonymousSeptember 25, 2011, 3:06 AM

    The protocol very useful. Thankyou very much for it. I have a query regarding the calculation part,

    "Take the number of micromoles tyrosine equivalents released obtained from the slope equation and multiply it by the total volume of the assay in mls, which in our case is 11mls. Divide this value by three other quantities: the time of the assay, which we ran for 10 minutes, the volume of enzyme used in the assay, which was varied (let's use 1ml), the volume of milliliters used in colorimetric detection, which may differ based on your cuvette. We used ² mls."

    Here why is total volume of assay and volume of mililiters used in colorimetric detection is used in mathmatical expression? DŒs it matter if we take different volume of mililiters for use in colorimetric detection, as this may vary with the equipments we use for colorimetric detection. If this value is changed the result will be different, even for the same experiment?

    Thank you in advance for your kindness in clearing my confusion.
    Regards,
    Gopi.
    Reply

    Posted by: AnonymousSeptember 28, 2011, 11:33 PM

    1. If using 0.5 ml of enzyme instead of 1 ml in the assay , then you would have 0.5 in the denominator of the calculation. If ² ml of enzyme was used, then the value would be ².

    ². If different volumes (other than ² ml) of the final assay reaction volume are used to be read in the colorimetric detection, the different volume needs to be use in the denominator also. For instance, 1 ml instead of ² ml.
    Reply

    Posted by: Nathan A.October 5, 2011, 9:34 AM

    thanks a lot
    Reply

    Posted by: mary l.October 9, 2011, 10:32 AM

    dear sir,
    your protocol is very useful. my questionis- i have followed the above procedure fŒ determining the activity of crude bromelain extract. the activity i have got is 0.1²u/ml at 37degree celsius and at ph=7.5 . is it correct?
    Reply

    Posted by: AnonymousOctober 18, 2011, 8:35 AM

    The pH optimum for this enzyme is substrate specific. BRENDA indicates the when casein is used as a substrate, the pH optimum is 7 - 8.5, thus our neutral protease assay should be suitable for measuring the activity of Bromelain. However, we cannot really say what activity level to expect, since Sigma measures the activity of Bromelain with a chromogenic substrate and at a different pH (One unit will release 1.0 μmole of p-nitrophenol from Nα-Z-L-lysine p-nitrophenyl ester per min at pH 4.6 at ²5 °C.).
    Reply

    Posted by: Nathan A.October 18, 2011, 12:59 PM

    Hello there,

    I have question pertaining to casein stock solution of 0.65%. I observed that over time there was this slimy-looked precipitate formed. Is it normal and okay to be used for assay? May I know how to avoid this precipitation?

    - Jameel
    Reply

    Posted by: Jameel J.February 5, 2012, 4:50 AM

    Hello Jameel,

    The Casein substrate (0.65% w/v) dŒs not readily go into solution and I would not necessarily expect it to stay in solution for an extended time period (Heat gently with stirring to 80-85 deg. C. for approximately 10 minutes until a homogeneous dispersion is achieved. Our QC department prepares this directly before use in the assay and has not stored it or evaluated the stability over time. You could might be able to take it back through the heating step to get it into solution, but I do not know what effect this would have on the assay values. I would recommend preparing it fresh again before the assay.
    Reply

    Posted by: Nathan A.February 8, 2012, 3:56 PM

    Hello Nathan,

    Thank you for your comment.

    -Jameel
    Reply

    Posted by: Jameel J.March 27, 2012, 5:42 AM

    Hi, there, I have got some questions:
    1. Could you tell me what the difference of alpha and beta casein
    ². What kind of casein are you using for the assay?
    3. Do you know some metalloproteases only degrade beta casein but not alpha casein?
    - Randy -
    Reply

    Posted by: AnonymousMarch 15, 2012, 3:20 PM

    Hello,
    1.) The link following includes a table that indicates the differences in the physical properties of the various Casein isoforms: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/c7078pis.Par.0001.File.tmp/c7078pis.pdf
    ².) The Casein (C7078) which is utilized in this assay has not been characterized as to what percentage of each of the different Casein isoforms are present. The approximate Casein composition of milk is (in g/ml): α-s1 (1²-15), α-s² (3-4), β (9-11), and κ (²-4). As our our method of preparation for this product dŒs not differentiate between the various Casein isoforms, they should be present in approximately the same ratios.

    3.) We are not aware of any metalloproteinases that can differentiate between alpha- and beta-Caseins. However, you may wish to search the literature regarding this property of MMP's to verify this.

    Nathan
    Reply

    Posted by: Nathan A.March 16, 2012, 10:13 AM

    Dear madam,
    I am doing research on microbial proteolytic activity in the spoilage of vacuum packaging cooked shrimp.Do you know how can i get the crude protease extract.

    I try to use 50 mM Potassium Phosphate Buffer to mix meat with four volumes in a stomacher. The mictures were then centrifuged (10000g for 30 min at 4°C),and the
    meat supernatants to be my crude protease extract. then follow your video.

    but have not good results.My absorbance values are very low.

    Dr. madam can help me to resolve them?


    Reply

    Posted by: AnonymousMarch 18, 2012, 4:37 PM

    In an attempt to troubleshoot your results, I would initially pose the following questions:

    1. Was a satisfactory standard curve obtained utilizing the Tyrosine standards?

    ². Did you utilize a positive enzyme control in order to confirm that the assay was working correctly? A product such as P5147 (Protease from Streptomyces griseus) would be suitable.

    3. You did not indicate the actual protein concentration of the supernatant you are assaying. You may need to make it more concentrated.

    4. This assay is designed to measure neutral protease activity. The non-specific protease needs to have an optimal activity in the range of pH 7.0 - 8.0. If the protease activity which you are trying to measure has an optimal activity which is far outside of this range, the assay may not be suitable.

    Nathan
    Reply

    Posted by: Nathan A.March 19, 2012, 2:29 PM

    1. Was a satisfactory standard curve obtained utilizing the Tyrosine standards?

    Yes, the tryisine standards curve have be set up.

    ². Did you utilize a positive enzyme control in order to confirm that the assay was working correctly? A product such as P5147 (Protease from Streptomyces griseus) would be suitable.

    NO,i have not pure Protease to try.
    3. You did not indicate the actual protein concentration of the supernatant you are assaying. You may need to make it more concentrated.
    Yes, i did not indicate the actual protein concentration of the supernatant, so do i prolong the reaction time that supernatant react on casein at 37°C ?
    4. This assay is designed to measure neutral protease activity. The non-specific protease needs to have an optimal activity in the range of pH 7.0 - 8.0. If the protease activity which you are trying to measure has an optimal activity which is far outside of this range, the assay may not be suitable.

    it's nice advice. thank you very much

    Reply

    Posted by: AnonymousMarch 21, 2012, 1:02 AM

    Is it possible to centrifuge enzyme mixture instead of using 0.45 um polyethersulfone syringe filter?
    Reply

    Posted by: AnonymousMarch 19, 2012, 4:47 AM

    HI It was so useful for me but i have a question can i check the activity of protease(crude extract) produced by a fungus???
    Reply

    Posted by: AnonymousMarch 19, 2012, 10:24 AM

    1.This assay is designed to measure neutral protease activity. The non-specific protease needs to have an optimal activity in the range of pH 7.0 - 8.0. If the fungal protease which you are trying to measure has an optimal pH which is far outside this range, the assay may not be suitable for their application.

    ². I would also recommend you utilize a positive enzyme control in order to confirm that the assay is correctly working. A product such as P5147 (Protease from Streptomyces griseus) would be suitable.
    Reply

    Posted by: Nathan A.March 19, 2012, 2:30 PM

    Dear madam
    i want to use the azocasein (sigma A²765 ) as a Substrate to test different bacteria's Non-specific Protease Activity in food.
    Could you tell me what the difference of the azocasein and the casein as a Substrate .
    thank you very much
    Reply

    Posted by: AnonymousMarch 25, 2012, 11:52 AM

    Hello,
    Azocasein is not suitable for use in the neutral protease assay, as it is a chromogenic substrate. This link will display an assay for Azocasein-http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/General_Information/azocasein_substrate_trypsin.Par.0001.File.tmp/azocasein_substrate_trypsin.pdf
    Reply

    Posted by: Nathan A.March 26, 2012, 11:31 AM

    Is it possible to centrifuge enzyme mixture instead of using 0.45 um polyethersulfone syringe filter? Also is it possible to use any type of syringe filter other than polyethersulfone?
    Reply

    Posted by: AnonymousApril 5, 2012, 2:12 AM

    We have not found centrifugation of the stopped reaction cocktail to be suitable in providing a clarified sample for spectrophotometric readings. The syringe filter dŒs not specifically have to be polyethersulfone.
    Reply

    Posted by: Nathan A.April 5, 2012, 11:48 AM

    Thank you for your reply Nathan. Yesterday, before your reply, I tried centrifugation and got a clarified sample. Is there any other disability of centrifugation other than this. Today, I will both centrifuge and filter the mixture and compare the results.
    Reply

    Posted by: AnonymousApril 6, 2012, 2:20 AM

    Thank you for your reply Nathan.
    Yesterday, before your reply, I tried centrifugation and got a clarified sample. Is there any other disability of centrifugation other than this. Today, I will both centrifuge and filter the mixture and compare the results.
    Reply

    Posted by: AnonymousApril 6, 2012, 2:19 AM

    As long as a linear plot is observed with the standards and you are able to replicate the same activity with multiple samples, it should work. The sample replication would be the largest question mark.
    Reply

    Posted by: Nathan A.April 6, 2012, 1:30 PM

    Dear Nathan,

    In the assay, I have tested the positive control, Bromelain from pineapple stem, which we purchased from SIGMA. On the bottle it indicates 3-7 U/mg. So, I just chose to weight 40 mg and dissolve it in 500 ml of the suggested enzyme diluent buffer. In the end, I did get specific activity at ~5.1 U/mg after measuring the protein concentration. Indeed, the value is within the range, however, in term of U/ml it was not as expected; 0.4 U/ml. Instead I got 0.0537 U/ml. Exactly, which one do I have to refer to?

    Thank you in advance.

    -Jameel
    Reply

    Posted by: Jameel J.April 30, 2012, 10:45 AM

    Hello,
    You won't be able to match the label units because Sigma dŒs not use the Non-specific protease assay (casein) to measure the activity of this enzyme. The Bromelain assay which is utilized by Sigma uses the chromogenic substrate Na-Z-L-lysine p-nitrophenyl ester. This is a link to that assay-www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/bromelaincsrd.Par.0001.File.tmp/bromelaincsrd.pdf
    Reply

    Posted by: Nathan A.April 30, 2012, 2:35 PM

    Dear madam,
    My control is also turning blue in color after the addition of FC. Could you please help me over this.

    Thanks,

    Naveen
    Reply

    Posted by: Naveen M.June 9, 2012, 11:21 PM

    Dear Madam,

    In the plate assays, i see that my organisms are protease positive. However in broth medium protease activity is not been observed.

    Thanks for all the help.

    Naveen
    Reply

    Posted by: Naveen M.June 9, 2012, 11:31 PM

    The Folin & Ciocalteu²17;s based Protease assay should be compatible with cell derived from either type of culture. DŒs the liquid culture medium contain any component that may interfere with the assay? Not knowing the components of the medium it is hard to judge. I would suggest that the cells should have been extracted from the medium by centrifugation prior to analysis, eliminating any interferant in the media. Please describe how the cells were treated and the procedure by which the protease assay was carried out.
    Reply

    Posted by: Nathan A.June 14, 2012, 5:41 PM

    Madam, we are using trypsin (1000 BAEE unit/mg) for cassein assay. However, we are not getting any color difference between test blank and the test samples. I have tried using trypsin solutions of activity .², .5 per ml. addition of TCA produces white ppt. but no turbidity on adding sodiumcarbonate as mentioned in the assay. Pl advice
    regards
    sharda
    Reply

    Posted by: Dr SHarda P.June 13, 2012, 1:52 PM

    The Trypsin you are using (1,000 BAEE units/mg) is assayed using the chromogenic substrate BAEE and these units are not equivalent to the units utilized in the neutral protease assay. Sigma-Aldrich has not evaluated the activity for any of our Trypsin products using the neutral protease assay. I would expect that you would need to add a much larger amount of Trypsin in the neutral protease assay then the 0.1 - 0.² unit/ml stock solution of protease. The second issue would be that Trypsin only cleaves on the C-terminal side of Lysine and Arginine. There may not be a large number of these sites which are present in casein and are freely accessible to Trypsin. You may wish to use the Sigma-Aldrich BAEE assay which is available at the following link:
    http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-trypsin.html
    Reply

    Posted by: Nathan A.June 14, 2012, 5:43 PM

    good morning i want to ask about Bromelain 3-7 units/mg protein, i bought it but i need extra information for example the activity of this is 3-7 units/mg protein but i would like to know its conversion to comercial units like standar qualities (FIP unit/mg, CDU/mg, GDU/g)..

    I really appreciate for your time and wait for your answer...
    Reply

    Posted by: viviana s.August 19, 2012, 12:30 PM

    Unfortunately, Sigma dΠnot evaluate the activity of our Bromelain enzyme products using the Non-specific protease assay (Casein as substrate). We actually measure the activity using the chromogenic substrate Na-Z-L-lysine p-nitrophenyl ester (see link below for assay). In addition, Sigma-Aldrich has not evaluated the activity of our Bromealin enzyme products in FIP, CDU, or GDU units, nor do we have a conversion factor on file for these type of units of activity. http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/bromelaincsrd.Par.0001.File.tmp/bromelaincsrd.pdf
    Reply

    Posted by: Nathan A.August 21, 2012, 12:46 PM

    Hello,

    I measured the activity of bacterial culture broth (after centrifugation). When I used different amount of the same enzyme (broth), I got different results. For example; with 0.05 ml of enzyme I read the absorbance as 0.090. However; with 1 ml of the same enzyme I determined the absorbance as 0.²90. What can be the reason? Interference?
    Reply

    Posted by: AnonymousSeptember 3, 2012, 7:33 AM

    There could be a few reasons for the results you are seeing. You could try the following tasks in order to identify what may be going on with your sample.

    1. Perform a spectral scan of the bacterial culture broth (after centrifugation) to to confirm that there is nothing present in the media which is absorbing at 660 nm.

    ². Perform the assay using the recommended sample volumes of 0.50 ml and 1.00 ml. Calculate the activity based upon the calculation provided in the assay.

    3. Perform the assay using a protease control such as product P5147 to insure that the assay and standard curve are functioning correctly.
    Reply

    Posted by: Nathan A.September 4, 2012, 1:58 PM

    Hello ,
    Could you please tell me how to prepare 1 micromole of tyrosine standard . I am finding difficulty in calculations please help me out and teach me .
    Reply

    Posted by: hafsa s.September 6, 2012, 11:01 AM

    The Tyrosine standard used in this assay should have a concentration of 1.1 mM and not 1 um. The 1.1 mM standard may be prepared by reconstituting 0.² mg of L-Tyrosine, Free Base, product number T3754, in 1 ml of purified water. Heat gently (do not boil) until the Tyrosine dissolves. This can also be prepared by reconstituting ²0 mg of Tyrosine into 100 ml of water.
    Reply

    Posted by: Nathan A.September 6, 2012, 2:38 PM

    But why we are not preparing 1 micromole what is the reason behind it please teach me . I am basically working on fungal protease and units are very low so I was thinking to prepare 1 micromole tyrosine .
    Reply

    Posted by: hafsa s.September 6, 2012, 11:25 PM

    The standard curve which is utilized in this assay (0.05 ml - 0.50 ml) of a 1.1 mM Tyrosine generates a standard curve in the range of 55 - umoles - 550 umoles). Sigma has done as low as 0.005 ml of the Tyrosine standard which would be equivalent to 5.5 umoles in the reaction mixture. Unfortunately, we have not evaluated the linearity of the assay, nor the detection limit below 5.5 umoles.
    Reply

    Posted by: Nathan A.September 7, 2012, 10:07 AM

    Thank you so much , could you please tell me is it possible if i will prepare 1mM of tyrosine standard curve.

    Posted by: hafsa s.September 8, 2012, 3:21 AM

    Can I ask something?
    Because I use this procedure to determine Protease from Aspergillus oryzae ≥500 U/g (product from Sigma) and I got the protease activity is 3500 U/g which is higher than 500 U/g
    I saw the Unit Definition which is One unit is the amount of enzyme which hydrolyzes 1 μmol of L-Ieucine-p-nitroanilide per minute, so can we use L-Tyrosine or L -leucine to conduct standard curve?
    Can I use this procedure to determine enzyme activity that is Protease from Aspergillus oryzae ≥500 U/g?
    Thank you very much for your' reply
    Best regard,
    Mary
    Reply

    Posted by: mary l.September 21, 2012, 6:23 AM

    Mary,
    Sigma-Aldrich has not evaluated the activity of product P6110 using the Non-Specific Protease Activity Assay (Casein). We obtain this product from Novozyme which measures the activity utilizing L-Leucine p-nitroanilide as the substrate. You will need to use the Novozyme assay to determine activity. You can obtain the assay by contacting the Sigma technical service team at techserv@sial.com.
    Reply

    Posted by: Nathan A.September 21, 2012, 1:26 PM

    Dear Nathan A.
    Thank you very much!
    Mary,
    Reply

    Posted by: mary l.September 21, 2012, 9:57 PM

    Dear Nathan A,
    Thank you very much for your reply.
    However,I check the label on the bottle of the product which is protease from Aspergillus oryae and I saw the Unit Definition One unit is the amount of enzyme which hydrolyzes 1 μmol of L-Tyrosine per minute which is difference with the definition on the website. My product form is power and have 3.6U/mg.
    My product is P6110, is right?
    Thank in advance for your reply!
    Mary,
    Reply

    Posted by: AnonymousSeptember 22, 2012, 10:44 PM

    Hello Mary,
    Could you please contact us directly at techserv@sial.com while referencing this video? We will be able to support you much quicker via email.
    Reply

    Posted by: Nathan A.October 1, 2012, 4:53 PM

    If we are using the kit to detect pepsin activity, what interfering substances should we be concerned about?
    Thank you!
    Janet
    Reply

    Posted by: Janet C.October 12, 2012, 3:58 PM

    If we are using the kit to detect pepsin activity, what interfering substances should we be concerned about?
    Thank you!
    Janet
    Reply

    Posted by: Janet C.October 12, 2012, 4:12 PM

    Sigma-Aldrich has not evaluated the use of this assay (Non-specific Protease Activity Assay - Casein) with any of our Pepsin enzyme products. I would not expect it to be suitable since the assay is performed at pH 7.5, which is a pH at which Pepsin would exhibit very little or no activity. The following method of assay is utilized by Sigma for our Pepsin enzyme products. http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/p7000enz.Par.0001.File.tmp/p7000enz.pdf
    Reply

    Posted by: Nathan A.October 17, 2012, 10:17 AM

    Dear Madam/ Sir,
    I am facing problem in calculations .please correct me
    Absorbance of (test - Control) / Absorbances of Standard X concentration of Standard.
    Is that is the tyrosine value that is equivalent liberated ?
    Why we are not using its molecular weight for division .
    Regards
    Hafsa
    Reply

    Posted by: hafsa s.October 19, 2012, 3:15 AM

    Dear Madam/ Sir,
    I am facing problem in calculations .please correct me
    Absorbance of (test - Control) / Absorbances of Standard X concentration of Standard.
    Is that is the tyrosine value that is equivalent liberated ?
    Why we are not using its molecular weight for division .
    Regards
    Hafsa
    Reply

    Posted by: hafsa s.October 19, 2012, 3:49 AM

    Hello,
    You do not need to utilize the molecular weight of Tyrosine in the calculation because you are obtaining the umoles of Tyrosine released from the standard curve which you set up.
    Reply

    Posted by: Nathan A.October 19, 2012, 1:27 PM

    Respected Mam / sir

    Kindly tell me that could i find proteolytic activity of protease enzyme using this protocol for the substrate whey protein isolate. Then could I come to know the proteolytic activity of protease from streptomyces griesus for whey protein substrate.
    Reply

    Posted by: Devi K.February 6, 2013, 2:27 AM

    Dear Nathan A.
    I tried to measure the bacterial activity from peat samples using this method. I found this video very informative, but I have some questions though:
    If I want to use a more acidic pH, what would be the best substrate to use?
    My test blank appeared very dark after the addition of Folin's reagent. I assume this is because the breakage of casein. I prepared the 0.65 % casein solution a day before assay. Can this be the reason?

    Mem
    Reply

    Posted by: Merja L.March 5, 2013, 4:16 AM

    Dear Nathan A.
    I tried to measure the bacterial activity from peat samples using this method. I found this video very informative, but I have some questions though:
    If I want to use a more acidic pH, what would be the best substrate to use?
    My test blank appeared very dark after the addition of Folin's reagent. I assume this is because the breakage of casein. I prepared the 0.65 % casein solution a day before assay. Can this be the reason?

    Mem
    Reply

    Posted by: Merja L.March 5, 2013, 5:41 AM

    Well, personally I would recommend you to reduce the concentration of your enzyme . I was also facing the same problem . Try this , I hope it will work.
    Hafsa Sattar
    Reply

    Posted by: hafsa s.March 8, 2013, 5:40 AM

    hi there i wanted to ask if there is any other alternative for calcium acetate??? could i used calcium chloride or oxalate instead?? i cant seem to find calcium acetate anywhere... plz do help.....
    Reply

    Posted by: Emily D.April 27, 2013, 10:16 PM

    Video is really informative. I need to do this assay to determine the activity of some of the protease enzymes I have. The optimum temperature and pH of my enzymes are 32 degrees C and pH 5.0, (so I am assuming I have to use these conditions to test my enzymes ?)
    - In the assay, you have used potassium phosphate buffer at pH 7.5. Would you recommend the same buffer at pH 5.0 for me to use?

    - Enzyme diluent solution used: 10 mM Sodium Acetate Buffer with 5mM Calcium Acetate, pH 7.5, at 37°C. Why do we need to add calcium acetate to sodium acetate buffer? And can I use the same at pH 5.0?
    Reply

    Posted by: Divya R.April 27, 2014, 11:16 PM

    Dear Nathan, the video is very useful, :-)
    but i dont really understand about preparing FC reagent. If the initial concentration of FC reagent is 2 N, then you dilute it with water 1:3, the final concentration should be 0.5 N instead of 0.5 mM. Could you please explain to me about the calculation ? Thank you :-)
    Reply

    Posted by: rani y.July 13, 2014, 1:41 AM

    Hello,
    The written description is correct: Step 4. 0.5 M Folin & Ciolcalteu’s, or Folin’s Phenol Reagent but the video frame is wrong. I apologize for the confusion. We'll work to get the video corrected.
    Best,
    Nathan
    Reply

    Posted by: Nathan A.July 17, 2014, 9:10 AM

    Dear Nathan

    This video is very useful and informative, i had tried this method in my laboratory to detect pretease activity in commercial enzym. I found that the activity is poorer than company claim. I dilute them to 0.1-0.2 U/gram. But the result is very bad and the colour didn't change. Can you help me to solve the problem? I think my procedure had been correct. Thank you.
    Reply

    Posted by: Ine I.August 14, 2014, 11:38 PM

    Dear Nathan,

    I diluted the samples to 0.1-0.2 Units/ml, but the end result was cloudy (it seems like when you added TCA). Can you tell me what the matter with that? Must all solution be fresh? thank you

    Reply

    Posted by: Ine I.August 20, 2014, 3:41 AM

    Hello Nathan, i wanna ask please..what does "volume used in Calorimetric Determination" mean? Is it volume of filtrate we add to Na2CO3 or volume of sample we add in cuvvette when meassuring absorbance ? Thank u :-)
    Reply

    Posted by: rani y.October 5, 2014, 10:33 PM

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