Name: Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis (Video) | JoVE Journal
Uploaded: 2025-09-26T13:00:00.000+00:00
Duration: 6 min 14 s
Description: Learn to separate insoluble cell wall and membrane proteins from Mycobacterium tuberculosis using preparative isoelectric focusing and molecular weight separation. This method enables direct proteomic and immunological analysis for TB diagnostics.

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5 minutes

This work is fully supported by the DST SERB grant of Dr. K. R. Uma Devi. Dr. B. Ramalingam was funded by the DBT-Ramalingaswamy fellowship.

This study has complied with the relevant governmental guidelines and was approved by the Institutional Scientific Advisory Committee and Ethical Committee. Informed consent was obtained from the study population before the samples were drawn.
1. Culturing of M. tuberculosis
<ol>
	<li>Transfer approximately 10 M. tuberculosis colonies from Lowenstein-Jensen slants to 2 mL of 7H9 broth medium (suspend 2.35 g of Middlebrook 7H9 Broth Base in 400 mL of distilled water, 50 mL of Oleic Albumin Dextrose Catalase [OADC] supplement, 0.5 g of Tween 80, and make up to 500 mL) 
	NOTE: M. tuberculosis is a Risk Group 3 pathogen; it should be handled in a biological safety cabinet (BSC) in the BSL3 facility. Appropriate personal protective equipment (PPE) must be used.</li>
	<li>Disperse the cells using glass beads under sterile conditions in a bottle shaker at 15 rpm for 1 min.</li>
	<li>Transfer the bacterial suspension to 10 mL of 7H9 broth medium in a McCartney bottle and incubate at 37 &#176;C for 2 weeks in a shaker incubator at 30 rpm.</li>
	<li>Scale up the log-phase culture to 200 mL of 7H9 broth medium and grow in a shaker culture for 4 weeks at 30 rpm.</li>
	<li>Further, scale up the culture in a 1-L flask containing 500 mL of 7H9 broth medium and grow as a shaker culture at 30 rpm for 4 weeks at 37 &#176;C.</li>
	<li>Harvest the mycobacteria by centrifuging at 1741 g for 30 min.</li>
</ol>
2. Preparation of whole cell lysate proteins
<ol>
	<li>Subject the isolated bacterial pellet to sonication following a standard protocol.</li>
	<li>Prepare the breaking buffer with the following composition: 20 mM Tris with 8.5% (8.5 g per 100 mL) NaCl.</li>
	<li>Adjust the final pH to 7.4 with concentrated HCl.</li>
	<li>Add freshly prepared protease inhibitors to the breaking buffer with the following concentrations (details of protease inhibitors to be added are listed in Supplementary Table 1)</li>
	<li>Treat M. tuberculosis bacilli (100 mL packed volume and weighed) for every 2 g of the bacterial cell pellet, add 1 mL of breaking buffer with lysozyme (1 mg/5 mL of cell suspension) and incubate for 15 min at 37 &#176;C with gentle stirring.</li>
	<li>Suspend the bacilli in twice its volume of breaking buffer.</li>
	<li>Add 0.04% (wt/vol) sodium azide and 0.05% Tween 80 to the buffer.</li>
	<li>Disrupt the bacterial cells in the breaking buffer using a sonicator with a cycle of 9 s on and 9 s off for 30 min at 40% amplitude, keeping the suspension on ice to prevent protein denaturation.</li>
	<li>Centrifuge the prepared lysate at 3438 g for 15 min at 4&#160;&#176;C to remove unbroken cells.</li>
	<li>Concentrate the supernatant using centrifugal concentrators (3 kDa cutoff- 5 mL).</li>
	<li>Determine the protein concentration in the supernatant using the bicinchoninic acid (BCA) protein quantification assay using BSA protein as standard. 
	NOTE: In SDS-PAGE analysis, load 50 &#181;g protein for analysis.</li>
</ol>
3. Isolation of cell wall and cell membrane proteins by ultracentrifugation
<ol>
	<li>Isolate cell wall, cell membrane, and cytosolic proteins from the whole cell lysate following the protocol by Raja et al.9. as described below.</li>
	<li>Centrifuge the whole cell lysate at 13751 &#215;&#160;g at 4 &#176;C for 30 min, separating the cell wall proteins as a pellet.</li>
	<li>Subject the supernatant to ultracentrifugation at 100,000&#160;&#215;&#160;g at 4 &#176;C for 4 h using an ultracentrifuge, separating the cell membrane proteins as a pellet and leaving the cytosolic proteins in the supernatant.</li>
	<li>Estimate protein concentrations in the cell wall and cell membrane fractions using the BCA protein assay. 
	NOTE: In SDS-PAGE analysis, load 50 &#181;g protein for analysis. Separated cell wall and cell membrane are stored in a -80 &#176;C freezer till further analysis.</li>
</ol>
4. Preparative liquid-phase isoelectric focusing (Rotofor [IEF apparatus])
<ol>
	<li>Solubilize the cell wall and cell membrane proteins isolated by ultracentrifugation in an IEF buffer containing 8 M urea, 1 mM DTT, 5% glycerol, 2% digitonin.</li>
	<li>Add 2% ampholytes (pH 3.0 to 10.0 and pH 4.0 to 6.0 at a 1:4 ratio)</li>
	<li>Fractionate the solubilized proteins using a liquid IEF system maintained at 4 &#176;C with a cooling water bath.</li>
	<li>Conduct the IEF separation according to the manufacturer&#39;s instructions, applying a constant power of 12 W until the voltage stabilizes (between 1300 V and 1600 V).</li>
	<li>Continue the IEF run for an additional 30 min after voltage stabilization, with a total run time of approximately 5-6 h.</li>
	<li>Collect the individual IEF fractions using a vacuum pump and determine their pH values.</li>
	<li>Subject the separated fractions (50 &#181;g) to SDS-PAGE and visualize the proteins using Coomassie Brilliant Blue (CBB) or silver staining. Store the separated IEF fractions in a -80 &#176;C freezer till further analysis.</li>
</ol>
5. Preparative SDS-PAGE and whole gel elution
<ol>
	<li>Mix the IEF-separated cell wall and cell membrane fractions with 6x SDS-PAGE sample buffer and heat at 95 &#176;C for 5 min before SDS-PAGE analysis.</li>
	<li>Separate the protein fractions in the second dimension using 16 cm 20 cm polyacrylamide gels consisting of a 12.5% resolving gel and a 4% stacking gel.</li>
	<li>Use a single 13-cm-long sample well for loading the sample.</li>
	<li>Conduct electrophoresis at a constant current of 50 mA/gel until the dye front reaches the bottom of the gel.</li>
	<li>Equilibrate the gel in elution buffer (60 mM Tris, pH 9.4; 40 mM 3-(Cyclohexylamino)-1-propanesulfonic acid [CAPS]) for 10 min after the electrophoresis run.</li>
	<li>Transfer the gel to a whole gel eluter apparatus as described by the instrument manufacturer.</li>
	<li>Elute the proteins from the gel by running the eluter at a constant current of 250 mA for 1 h.</li>
	<li>Collect approximately 30 protein fractions, each with a volume of 3 mL, from each gel using a vacuum pump.</li>
	<li>Quantify the protein concentration in the eluted fractions using the BCA protein assay.</li>
	<li>Subject 10 &#181;g of the eluted fractions to SDS-PAGE analysis and visualize by Coomassie brilliant blue (CBB) staining. 
	NOTE: Separated whole gel eluted fractions were stored in the -80 &#176;C freezer till further analysis.</li>
</ol>

<ol><li>Seshadri, C., Turner, M. T., Lewinsohn, D. M., Moody, D. B., Van Rhijn, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipoproteins+are+major+targets+of+the+polyclonal+human+T+cell+response+to+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Lipoproteins are major targets of the polyclonal human T cell response to Mycobacterium tuberculosis.</a> J Immunol. 190 (1), 278-284 (2013).</li>
<li>Kunnath-Velayudhan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+antibody+responses+to+the+Mycobacterium+tuberculosis+proteome%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Dynamic antibody responses to the Mycobacterium tuberculosis proteome.</a> Proc Natl Acad Sci U S A. 107 (33), 14703-14708 (2010).</li>
<li>Drage, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TLR2+and+its+co-receptors+determine+responses+of+macrophages+and+dendritic+cells+to+lipoproteins+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Cell+Immunol%22%5BJournal%5D)">TLR2 and its co-receptors determine responses of macrophages and dendritic cells to lipoproteins of Mycobacterium tuberculosis.</a> Cell Immunol. 258 (1), 29-37 (2009).</li>
<li>Wallin, E., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+analysis+of+integral+membrane+proteins+from+eubacterial%2C+archaean%2C+and+eukaryotic+organisms%5BTitle%5D)+AND+%22Protein+Sci%22%5BJournal%5D)">Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms.</a> Protein Sci. 7, 1029-1038 (1998).</li>
<li>Almeida, J. G., Preto, A. J., Koukos, P. I., Bonvin, A. M. J. J., Moreira, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+proteins+structures%3A+A+review+on+computational+modeling+tools%5BTitle%5D)+AND+%22Biochim+Biophys+Acta+Biomembr%22%5BJournal%5D)">Membrane proteins structures: A review on computational modeling tools.</a> Biochim Biophys Acta Biomembr. 1859 (10), 2021-2039 (2017).</li>
<li>Finkelstein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structures+of+membrane+proteins%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structures of membrane proteins.</a> Nature. 511 ((Suppl 7509)), 21(2014).</li>
<li>Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+for+the+purification+of+membrane+proteins%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Strategies for the purification of membrane proteins.</a> Methods Mol Biol. 1485, 389-400 (2017).</li>
<li>Von Jagow, G., Link, T., Schager, H. Purification Strategies for Membrane Proteins. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVon+Jagow%2C+G+%22A+Practical+Guide+to+Membrane+Protein+Purification%22">A Practical Guide to Membrane Protein Purification</a>. , Academic Press. San Diego, CA. (1994).</li>
<li>Raja, A., Uma Devi, K. R., Ramalingam, B., Brennan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoglobulin+G%2C+A%2C+and+M+responses+in+serum+and+circulating+immune+complexes+elicited+by+the+16-kilodalton+antigen+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Clin+Diagn+Lab+Immunol%22%5BJournal%5D)">Immunoglobulin G, A, and M responses in serum and circulating immune complexes elicited by the 16-kilodalton antigen of Mycobacterium tuberculosis.</a> Clin Diagn Lab Immunol. 9 (2), 308-312 (2002).</li>
<li>Covert, B. A., Spencer, J. S., Orme, I. M., Belisle, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+application+of+proteomics+in+defining+the+T+cell+antigens+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis.</a> Proteomics. 1 (4), 574-586 (2001).</li>
<li>Deenadayalan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoproteomic+identification+of+human+T+cell+antigens+of+Mycobacterium+tuberculosis+that+differentiate+healthy+contacts+from+tuberculosis+patients%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Immunoproteomic identification of human T cell antigens of Mycobacterium tuberculosis that differentiate healthy contacts from tuberculosis patients.</a> Mol Cell Proteomics. 9 (3), 538-549 (2010).</li>
<li>Sinha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunogenic+membrane-associated+proteins+of+Mycobacterium+tuberculosis+revealed+by+proteomics%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.</a> Microbiology. 151 (7), 2411-2419 (2005).</li>
<li>Deenadayalan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Integrated+approach+for+proteomic+and+immunological+analysis+of+cell+wall+and+cell+membrane+proteins+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Med+Res+Arch%22%5BJournal%5D)">An Integrated approach for proteomic and immunological analysis of cell wall and cell membrane proteins of Mycobacterium tuberculosis.</a> Med Res Arch. 11 (11), (2023).</li>
</ol>

The authors have nothing to disclose.

The hydrophobic nature of cell wall and membrane proteins presents significant experimental challenges in their solubilization and separation. In the abovementioned protocol, we described a novel two-dimensional electrophoresis-based separation method to effectively isolate these hydrophobic proteins. In this protocol, we initially separated the membrane and cell wall proteins based on their isoelectric points using the liquid IEF system. To solve the solubilization problem, we used a high concentration of chaotropic age...

Immunological studies have demonstrated that in addition to soluble proteins, which are easy to purify, the human immune response to pathogens such as M. tuberculosis is also directed hydrophobic protein present in the cell wall1 and cell membrane proteins2 of Mycobacterium tuberculosis. These cell wall and membrane proteins play a crucial role in immunological control against many pathogens, including tuberculosis. Some of these proteins are reported as immunodominant antigens that mount T and B cell response. In contrast, other proteins are identified as molecular signatures of pathogens that are recognized to initiate an innate immune response, such as pathogen-associated molecular patterns (PAMPs) by immune cells, thereby initiating an immune response. For example, Mycobacterial lipoproteins such as LprG, LpqH, LprA, and PhoS1 are recognized by Toll-like receptor 23. 
The hydrophobic nature of cell wall and membrane proteins presents significant technical challenges in terms of their solubilization and separation. Studies have shown that membrane proteins constitute approximately 20% to 30% of the coding regions of all organisms, including mycobacteria4,5. Due to their intrinsic nature and technical challenges, cell wall and membrane proteins are more difficult to solubilize and isolate compared to soluble proteins. As a result, only 2% of membrane protein structures have been reported6. Several factors contribute to the difficulty of isolating these proteins. The primary challenge is their low abundance within the cell, which complicates the isolation of individual proteins for further characterization7. Additionally, these proteins tend to aggregate, further complicating their solubility. This issue can be addressed by adding detergents to aid in solubilization. However, some proteins may still form aggregates even in the presence of detergents, in which case chaotropic agents and reducing agents can be used to improve solubility.
The use of detergents for solubilization can interfere with subsequent separation methods. For instance, charged detergents cannot be used with ion exchange chromatography, and all detergents can disrupt hydrophobic interaction chromatography8. Therefore, selecting a detergent compatible with the chosen separation method is critical. Another important step in cell wall and membrane protein isolation is the removal of detergents used for solubilization, as these can interfere with subsequent functional analyses of the proteins.
We have developed a robust methodology that overcomes many of the challenges associated with the analysis of membrane proteins, particularly the hydrophobic proteins from the cell wall and cell membrane. A key challenge in studying these proteins is their solubilization, as their hydrophobic nature makes them difficult to extract and analyze. The methodology described here employs a specially formulated isoelectric focusing (IEF) buffer, which efficiently solubilizes cell wall and cell membrane proteins. This solubilization step is critical as it enables the subsequent analysis of these hydrophobic proteins.
An additional advantage of this method is its ability to reduce the complexity of the protein mixture. By employing two complementary separation techniques-based on the isoelectric point (pI) and molecular weight of proteins-this methodology efficiently separates cell wall and cell membrane proteins into simplified fractions. Each fraction typically contains only one to three proteins, significantly enhancing the resolution and facilitating downstream characterization.
This methodology, developed using Mycobacterium tuberculosis cell wall and cell membrane proteins as a model system, effectively addresses the challenges associated with isolating and analyzing hydrophobic proteins. It provides a reliable approach for the isolation, separation, and characterization of these complex protein fractions, advancing the ability to study them in detail.

<table><tbody><tr><td>7H9 broth&amp;nbsp;</td><td>Himedia&amp;nbsp;</td><td>M198</td><td></td></tr><tr><td>40% Bio-Lyte 3/10 Ampholyte&amp;nbsp;</td><td>BIO-RAD</td><td>1631113</td><td></td></tr><tr><td>BCA protein assay&amp;nbsp;</td><td>MERCK</td><td>BCA1</td><td></td></tr><tr><td>Bio-Lyte 5/7 Ampholyte&amp;nbsp;</td><td>BIO-RAD</td><td>1631152</td><td></td></tr><tr><td>centrifugal concentrators 3 kDa</td><td>MERCK</td><td>Z629456</td><td></td></tr><tr><td>Digitonin.</td><td>MERCK</td><td>D141</td><td></td></tr><tr><td>Dnase</td><td>MERCK</td><td>11284932001</td><td></td></tr><tr><td>DTT</td><td>MERCK</td><td>D9779</td><td></td></tr><tr><td>EDTA</td><td>MERCK</td><td>E9884</td><td></td></tr><tr><td>EGTA</td><td>MERCK</td><td>E3889</td><td></td></tr><tr><td>Glycerol</td><td>MERCK</td><td>G5516</td><td></td></tr><tr><td>Leupeptin</td><td>MERCK</td><td>E18</td><td></td></tr><tr><td>Pepstatin A</td><td>MERCK</td><td>P5318</td><td></td></tr><tr><td>PMSF</td><td>MERCK</td><td>78830</td><td></td></tr><tr><td>Rnase</td><td>MERCK</td><td>10109134001</td><td></td></tr><tr><td>Rotofor&amp;nbsp;</td><td>BIO-RAD</td><td>170-2986</td><td>&amp;nbsp;isoelectric focusing (IEF) apparatus</td></tr><tr><td>Sonicator</td><td>Vibra-Cell, Sonics</td><td>VC 750</td><td></td></tr><tr><td>TLCK</td><td>MERCK</td><td>T7254</td><td></td></tr><tr><td>TPCK</td><td>MERCK</td><td>4376</td><td></td></tr><tr><td>urea</td><td>MERCK</td><td>U5378</td><td></td></tr><tr><td>Whole Gel Eluter apparatus&amp;nbsp;</td><td>BIO-RAD</td><td>1651250</td><td></td></tr></tbody></table>

separation and fractionation of cell wall and cell membrane proteins from mycobacterium tuberculosis for downstream protein analysis

Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis

Mycobacterium cell wall and membrane proteins, which play a central role in tuberculosis pathogenesis, were successfully separated using preparative Isoelectric Focusing (IEF) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Followed by gel elution, overcoming limitations in conventional methods for the separation of hydrophobic proteins. In this procedure, M. tuberculosis colonies were transferred from Lowenstein-Jensen slants into 2 mL of 7H9 broth, dispersed with glass beads, and incubated at 37 &#176;C for 2 weeks. Then, the culture was scaled up to 200 mL and grown in a shaker for 4 weeks. It was further upscaled to 1 L with 500 mL of 7H9 broth and grown for an additional 4 weeks. Grown Mycobacteria were pelleted by centrifugation at 1741 &#215;&#160;g for 30 min. For each 2 g pellet, 1 mL of breaking buffer was added, and the sample was sonicated. The lysate was centrifuged at 3436&#160;&#215;&#160;g for 15 min to remove unbroken cells, and the supernatant was concentrated. This supernatant (whole cell lysate) was centrifuged at 13751&#160;&#215;&#160;g for 30 min to pellet cell wall proteins. The remaining supernatant was ultra-centrifuged at 100,000&#160;&#215;&#160;g for 4 h to separate the cell membrane and cytosol. The isolated cell wall and membrane proteins were loaded onto a liquid preparative IEF system at 4 &#176;C and separated at 12 W until the voltage stabilized at 1400 V, which separates 20 fractions. These IEF fractions were further separated by preparative SDS-PAGE, and proteins were eluted using a whole gel eluter at 250 mA, resulting in 30 fractions. Through this protocol, we were able to identify novel M. tuberculosis cell walls and membrane-specific biomarkers, and it also shows potential for characterizing similar proteins in other pathogens.

Isolation of cell wall and cell membrane proteins by high-speed ultracentrifugation 
Cell wall and membrane proteins were isolated from whole cell lysates via ultracentrifugation as outlined in the protocol. SDS-PAGE analysis of the separated subcellular fractions-cell wall, cell membrane, and cytosol protein profiles of M. tuberculosis is shown in Figure 1.
Separation of cell wall proteins by preparative liquid-phase isoelectr...

This protocol separates insoluble cell wall and membrane proteins into simple fractions (1-5 proteins) using preparative isoelectric focusing (IEF) based on isoelectric point, followed by separation by molecular weight. The resulting fractions can be used directly for immunological and proteomic analysis without further purification.

Mycobacterium cell wall and membrane proteins, which play a central role in tuberculosis pathogenesis, were successfully separated using preparative ...

Our research focused on developing a streamlined method to separate insoluble cell wall and cell membrane proteins of Mycobacterium tuberculosis into simple fractions for direct proteomic and immunological assays. Advances in the electrophoretic techniques now allow complex proteins to be separated into fewer protein fractions, facilitating efficient mass spec-based isolation for functional proteomic and immunological applications. Key challenges include poor solubility of hydrophobic protein precipitating above hundred milligram and SDS-PAGE induced denaturation, which hinders downstream application needing native confirmation like enzyme activity assays.No current protocol systematically separates insoluble hydrophobic cell wall and cell membrane proteins into defined water-soluble fractions for direct proteomic and immunological analysis, which is key to understanding function and guiding interventions. Our future research will focus on developing T-cell antigen-based diagnostics and to evaluate their sensitivity, specificity, and performance across the TB population for an early, accurate TB detection with an aim to support better disease control. To begin, obtain the cell wall, cell membrane, and cytosolic protein samples from whole cell lysate of Mycobacterium tuberculosis bacilli.Solubilize the cell wall and cell membrane proteins in isoelectric-focusing buffer. Add 2%ampholytes between pH ranges of 3 to 10 and 4 to 6 in a ratio of one to four. Now fractionate the solubilized proteins in a liquid isoelectric-focusing system maintained at four degrees Celsius with a cooling water bath.Conduct isoelectric-focusing separation following the manufacturer's instructions by applying a constant power of 12 watts. Collect the individual isoelectric-focusing fractions with a vacuum pump. Determine the pH values of each fraction.Subject 50 micrograms of the separated fractions to SDS-PAGE and visualize the proteins using Coomassie Brilliant Blue or silver staining. Start by mixing the IEF-separated cell wall and cell membrane fractions with SDS gel electrophoresis sample buffer. Heat the mixture at 95 degrees Celsius for five minutes.Separate the protein fractions in the second dimension, using 16 by 20 centimeter polyacrylamide gels with a 12.5%resolving gel and a 4%stacking gel. Then use a single 13-centimeter-long sample well to load the sample onto the gel. Perform electrophoresis at a constant current of 50 milliamperes per gel until the dye front reaches the bottom of the gel.After electrophoresis, equilibrate the gel in elution buffer for 10 minutes. Transfer the gel to a whole gel eluter apparatus as per the instrument manufacturer's instructions. Run the eluter at a constant current of 250 milliamperes for one hour to elute the proteins from the gel, then use a vacuum pump to collect approximately 30 protein fractions, each measuring three milliliters, from the gel.Quantify the protein concentration in the eluted fractions using the bicinchoninic acid protein assay. Subject 10 micrograms of the eluted protein fractions to SDS-PAGE analysis and visualize the bands by Coomassie Brilliant Blue staining. Distinct protein banding patterns were observed in the first supernatant:cell wall, cell membrane, and cytosol fractions of Mycobacterium tuberculosis, confirming successful subcellular fractionation.The isoelectric focusing of cell wall proteins showed the highest protein concentration in fractions with pH below 2.5. SDS-PAGE of the cell wall IEF fractions revealed distinct protein bands, predominantly in Fractions 1 to 8. Cell membrane protein separation by IEF revealed major protein concentration peaks between pH 4 and pH 10, with the highest concentration occurring around pH 10.SDS-PAGE of the cell membrane IEF fractions showed diverse protein profiles with clearer bands in Fractions 1 to 9. Preparative SDS-PAGE separation of the IEF-resolved cell wall proteins produced fractions predominantly under 100 micrograms per milliliter. SDS-PAGE of the eluted cell wall protein fractions displayed a wide range of protein molecular weights, with more concentrated bands in Fractions 6 to 10.Preparative SDS-PAGE of cell membrane protein fractions revealed that over half of the fractions had protein concentrations between 50 and 199 micrograms per milliliter. SDS-PAGE of the eluted cell membrane protein fractions revealed concentrated bands in Lanes 1 to 8 and 13 to 16.

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Learn to separate insoluble cell wall and membrane proteins from Mycobacterium tuberculosis using preparative isoelectric focusing and molecular weight separation. This method enables direct proteomic and immunological analysis for TB diagnostics.

Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis (Video) | JoVE Journal

Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis

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