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Method Article

Gelatin Zymography to Detect Gelatinase Activity in Melanoma Cells

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DOI:

10.3791/63278

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March 9th, 2022

 ,  , 
1Department of Cell Pathology, Faculty of Biotechnology, University of Wrocław

In This Article

Summary

Metalloproteases (MMPs) are secreted by many cells, including malignant melanoma. MMP-mediated cleavage of extracellular matrix components leads to the increased invasive potential of these cells. Gelatin zymography, presented here, is a quantifying method for studying gelatinase activity manifested as a digested gelatin area on a polyacrylamide gel.

Abstract

Melanoma cells, having highly invasive properties, exhibit the formation of invadopodia—structures formed by tumor cells and responsible for the digestion of the surrounding extracellular matrix (ECM). Several metalloproteases (MMPs) are secreted by cells to hydrolyze ECM proteins. They are mainly secreted through structures known as invadopodia. ECM degradation is crucial for tumor cells while forming metastases as the cells heading towards blood vessels must loosen dense tissue.

One group of metalloproteases secreted by melanoma cells comprises the gelatinases, i.e., metalloproteases 2 and 9. Gelatinases cleave gelatin (denatured collagen), a few types of collagen (including type IV), and fibronectin, all structural components of ECM. This paper describes a gelatin zymography assay to analyze the gelatinase activity of melanoma cells. This approach is based on analyzing the extent of digestion of a substrate (gelatin) added to a polyacrylamide gel. Several advantages, such as simplicity, sensitivity, low cost, and semiquantitative analysis by densitometry, as well as the detection of both active and inactive forms of MMPs, make this assay valuable and widely used.

This protocol describes how to concentrate medium devoid of intact floating cells, cell debris, and apoptotic bodies. Next, it focuses on preparing polyacrylamide gel with gelatin addition, performing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), removing SDS, and staining of the gel to detect gelatin-free bands corresponding to the activity of gelatinases secreted by melanoma cells. Finally, the paper describes how to quantitatively analyze data from this assay. This method is a good alternative for estimating the gelatinase activity of melanoma cells to a fluorescent gelatin degradation assay, western blot, or enzyme-linked immunosorbent assays (ELISAs).

Introduction

Matrix metalloproteinases (MMPs) are a family of Zn2+-containing endopeptidases that cleave various ECM proteins and non-ECM proteins, such as growth factors, cell receptors, proteinases, and their inhibitors1,2,3,4. ECM-substrate specificity of MMPs is dependent on the peptide domains and motifs, as well as similarities in their sequences, thus defining their subgroups. There are, for example, collagenases digesting various types of collagens, as well as gelatin and aggrecan; gelatinases cleaving gelatins and collagens; and matrilysins or MT-MMPs that digest various ECM proteins5.

This paper focuses on two gelatinases: MMP-2 and MMP-9, as they can digest denatured collagen (gelatin) proteolytically, allowing the detection of their activity using a gelatin zymography assay3,6. Although MMP-2 and MMP-9 bear a strong structural resemblance, they do not have identical substrate specificity7. A C-terminal hemopexin-like domain of MMPs is responsible for recognizing substrate sequence8. Slight differences in their catalytic domains are responsible for the differences in the substrate selectivity of MMP-2 and MMP-9, e.g., MMP-2, unlike MMP-9, can cleave native type I collagen9. Nevertheless, their proteolytic activities can be unquestionably determined with gelatin zymography as they both can cleave gelatin3,6.

MMPs are already known to be involved in both physiological and pathological conditions. They were found to impact cell migration, invasion, spreading, and adhesion, thus impairing angiogenesis, inflammation, tumor progression, and metastasis10,11,12,13. As they take part in various important processes, they are extensively studied due to their high therapeutic or diagnostic potential14,15,16. MMP-2 (gelatinase A) occurs as a 72 kDa proenzyme whose prodomain binds Zn2+ in the catalytic site, leading to the inhibition of enzymatic activity9. MMP-2 can be activated through the cleavage of its zymogen's prodomain by MMP-14 (MT1-MMP), thrombin, and activated protein C17,18,19,20. Therefore, the mass of active MMP-2 is lower (~64 kDa). In contrast, MMP-9 (gelatinase B) is expressed as a ~92 kDa proenzyme and is activated by the cleavage of the N-terminal domain to obtain the 83 kDa protein. MMP-9 maturation results from a prodomain cleavage by serine proteases, other MMPs, and as a response to the oxidative stress21.

The progression and malignancy of melanoma are highly dependent on the ability of the tumor cells to digest ECM, as it is "a barrier" limiting the cells from progression and metastasis formation22. Cells need to first penetrate the basal membrane (BM) to enter the dermis, migrate toward blood vessels, adhere to vascular endothelium, and reach the blood. It has been shown that gelatinase expression was increased in different cancers and was correlated with increased invasion and migration and a worse prognosis for patients23,24. MMP-2 was highly expressed in melanoma cells, with its activation state correlated with progression25,26. MMP-9 was also found to accumulate in human skin tumors and melanoma cell lines27,28.

Due to the high correlation between the properties of MMPs with the invasiveness of melanoma cells, the availability of a simple, sensitive, low-cost, functional assay to determine their presence and activity is crucial for better understanding the biology of melanoma and designing new diagnostic techniques for their detection. This paper describes the gelatin zymography technique in detail, as it can be considered the best candidate for this purpose. This approach is based on SDS-electrophoresis under denaturing but nonreducing conditions, using polyacrylamide gels prepared with the addition of gelatin29,30. Although proteins, including MMP-2 and MMP-9, are denatured in the presence of SDS during electrophoresis, washing in buffer containing Triton X-100 causes their renaturation as the result of an SDS:Triton X-100 exchange31.

These renatured MMPs digest gelatin during gel incubation in an incubation buffer, which can be finally observed as clear zymolytic bands in the Coomassie Blue-stained gel5. The amount and the area of digested gelatin presented as transparent bands corresponding to the gelatinolytic activity of MMPs can be determined using both commonly used and open-source applications—ImageLab and ImageJ29,32. Although this method has many advantages, it also possesses some limitations mentioned in the discussion. This "step by step" protocol with notes and comments performed on the different melanoma cell lines should be sufficient for reproducibility and optimization to obtain representative results. Figure 1 presents the steps of the described procedure.

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Protocol

1. Cell culture medium collection and concentration

  1. Seed the melanoma cells (here A375, SK-MEL-28, Hs 294T, WM9, WM1341D cell lines) into tissue-culture 75 cm2 flasks in complete medium [Dulbecco's modified Eagle's medium-high glucose with reduced concentration (1.5 g/L) of NaHCO3, supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 1% (v/v) L-Glutamine, and 1% (v/v) Antibiotic-Antimycotic].
  2. Culture the cells under standard conditions (5% CO2, 37 °C).
  3. After the cells reach 80-90% confluence, aspirate and discard the supernatant and wash the flasks 3 times with serum-free medium or PBS warmed to 37 °C to remove residual medium in the culture flask.
    NOTE: The supernatant (complete medium) should be removed completely before adding the serum-free medium. FBS contains various MMPs, which may lead to false-positive results and wrong data analysis and interpretation29.
  4. Add 10 mL of warm serum-free medium and incubate the cells at 37 °C in a humidified atmosphere of 5% CO2 for 48-72 h.
    NOTE: The duration of cell incubation in the serum-free medium may affect cell viability, as well as the amount of secreted proteins. Therefore, the duration of cell starvation must be optimized before performing zymography depending on the type of cell line. The trick is to choose when cells are most viable and secrete the highest amount of MMPs. All melanoma cell lines used here were cultured in a serum-free medium for 48 h without affecting viability. The condition of the cells was verified using a phase-contrast microscope, although an XTT or MTT cell viability assay can be performed.
  5. Collect the media from the cell cultures after 48-72 h and transfer them to 15 mL tubes. Keep the media constantly on ice to avoid protein degradation.
  6. Centrifuge the media for 20 min at 7,000 × g at 4 °C33.
    NOTE: Centrifugation is crucial for removing floating intact cells, cell debris, and apoptotic bodies, as they may be sources of active MMPs. Skipping this step could lead to false-positive results and wrong data analysis and interpretation. Optionally, media can be filtered through 0.22-µm filters to remove apoptotic bodies, large microvesicles, and cell debris34. It is important to centrifuge the media before transferring to -20 °C for storage, as freezing and thawing of cells may lead to their damage, thus releasing intracellular components35.
  7. Store the media at -20 °C if required before further analyses (STOP POINT 1).
  8. Thaw the media on ice and concentrate them to achieve the desired final concentrate volume using ultracentrifugal filter units with a 10-kDa cutoff (see the Table of Materials), according to the manufacturer's recommendation.
    NOTE: Here, the obtained concentration factor was approximately 20 times. As the molecular weight of the detected MMPs is 43-215 kDa, it is recommended to use ultracentrifugal filter units with 10 kDa Nominal Molecular Weight Limit (NMWL). Using higher NMWL (30 kDa) may result in losing the MMP molecules with lower molecular weight (e.g., 43 kDa collagenase MMP-1).
  9. Transfer the concentrated medium immediately to -80 °C and store at -80 °C (STOP POINT 2).
    NOTE: Fresh or thawed samples should be kept on ice during the next steps of the protocol to avoid protein degradation.
  10. Measure the concentration of proteins in collected and concentrated media using, e.g., the Bradford or BCA method, according to the manufacturer's recommendation (STOP POINT 3).

2. Preparation of SDS-PAGE separating and stacking gels with gelatin (1 mg/mL) for gelatin zymography

  1. Place the spacer plate with a short plate into a casting frame to form a cassette and place the frame on the casting stand.
    NOTE: The spacer plate with 0.75-1.5 mm integrated spacers can be used (here 1 mm). Ensure that the working surface is stable and perfectly leveled.
  2. Prepare 2.65 mg/mL of gelatin by weighing the appropriate amount of gelatin and dissolving it in ultrapure or sterile deionized H2O by warming the solution to 65 °C. Sterilize the solution using a 0.22-µm syringe filter. Cool the gelatin solution down to room temperature before preparing the separating gel.
    NOTE: The gelatin solution can be stored for 1 week at 4 °C. Long-term storage may cause bacterial contamination and the presence of proteolytic enzymes secreted by them36.
  3. Prepare 10 mL of 10% separating polyacrylamide gel by mixing 3.95 mL of 2.65 mg/mL gelatin, 3.3 mL of 30% acrylamide/bis-acrylamide, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 48 µL of 10% SDS, 80 µL of 50% glycerol, 70 µL of H2O, 48 µL of 10% APS, and 4.8 µL of TEMED.
    NOTE: CAUTION! Wear gloves and goggles when handling acrylamide/bis-acrylamide. APS (an activator) and TEMED (catalyst of gel polymerization) must be added at the end of gel preparation, after which it is important to work quickly and efficiently. CAUTION! TEMED must be added under a fume hood. This volume of the separating gel is sufficient to prepare two separating gels with a 1 mm spacer suited to the referenced electrophoresis apparatus (see the Table of Materials). If a lower percentage of the gel is required, recalculate the volumes of the components to obtain the desired percentage of acrylamide in the gel.
  4. Mix the 10% separating gel solution by inversion six to eight times, avoiding air bubble formation, and pour the solution into the gel cassette sandwich (step 2.1) up to 75% of the height of the short plate.
    NOTE: The thickness of the gel, the amount of loaded protein, and incubation time in the incubation buffer (see step 5.1.3 for composition) should be optimized to obtain areas of the gel with the fully digested gelatin bands separated for individual MMPs. Ultimately, this depends on the amount of MMPs secreted by a given cell type.
  5. Layer the top of the gel with 70% ethanol.
    NOTE: It is essential to remove bubbles and prevent the gel from drying out and forming contact with air, which is unsuitable for polymerization.
  6. Leave the gel for approximately 30 min at room temperature. When the polymerization is complete and a clear separation line between the gel mix and ethanol layers is visible, remove the ethanol layer carefully.
  7. Prepare 4% stacking gel (5 mL) by mixing 3.05 mL of H2O, 0.67 mL of 30% acrylamide/bis-acrylamide, 1.25 mL of 0.5 M Tris-HCl (pH 6.8), 50 µL of 10% SDS, 50 µL of 10% APS, and 5 µL of TEMED. Mix the 4% stacking gel solution by inversion six to eight times.
    NOTE: This volume is sufficient to prepare two stacking gels with a 1 mm spacer.
  8. Insert a comb on the top of the gel sandwich cassette immediately and fill it with the stacking gel solution.
  9. Incubate the stacking gel for approximately 30 min at room temperature to allow it to polymerize completely.
  10. Wrap the gels with moist paper towels, place them in a plastic bag, and keep the gels at 4°C for at least overnight but no longer than 1 week.
  11. Load the gel sandwich cassette into an electrode assembly, transfer it to the tank, and fill it with 1x SDS-PAGE buffer [250 mM Tris-HCl, 1.92 M glycine, 1% (w/v) SDS] before running the SDS-PAGE electrophoresis.
    NOTE: SDS-PAGE buffer can be stored at 4 °C for extended periods.
  12. Remove the comb from the gel.

3. Preparation of SDS-PAGE separating and stacking gels for total protein content determination

NOTE: The steps in section 3 are similar to the steps in section 2 describing gel preparation for gelatin zymography. Note that the composition of the gels is different. A gel for total protein content determination must not contain gelatin, as it will also be stained by Coomassie Brilliant Blue solution.

  1. Prepare 10 mL of 10% separating gel by mixing 3.34 mL of 30% acrylamide/bis-acrylamide, 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 100 µL of 10% SDS, 3.99 mL of H2O, 50 µL of 10% APS, and 20 µL of TEMED.
  2. Mix the solution by inversion, avoiding air bubble formation, and transfer the solution to the gel cassette sandwich up to 75% of the height of the short plate.
  3. Layer the top of the gel with ethanol and leave the gel for approximately 30 min at room temperature. Remove the ethanol.
  4. Prepare 5 mL of 4% stacking gel by mixing 650 µL of 30% acrylamide/bis-acrylamide, 1.25 mL of 1.5 M Tris-HCl (pH 8.8), 50 µL of 10% SDS, 2.99 mL of H2O, 25 µL of 10% APS, and 12.5 µL of TEMED.
  5. Place a comb on the top of the gel sandwich cassette and fill it with stacking gel solution.
  6. Leave the gel for approximately 30 min at room temperature.
    NOTE: The gel can be prepared just before SDS-PAGE electrophoresis or stored for a few days at 4 °C. For gel storage, wrap the gel in a wet paper towel and place it in a plastic bag to keep it moist.
  7. Put the cassettes with the gels in the tank, load the gel sandwich cassette into an electrode assembly, and transfer the assembly to a tank with 1x SDS-PAGE buffer [250 mM Tris-HCl, 1.92 M glycine, 1% (w/v) SDS] before running the SDS-PAGE electrophoresis.
  8. Remove the comb from the gel.

4. Sample loading and electrophoresis running

  1. Prepare each SDS-PAGE sample for zymography by mixing a 1:1 ratio of 3-20 µg (here 10 µg) of concentrated medium with 2x nonreducing loading buffer [10 mM Tris pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol, 0.03% (w/v) bromophenol blue] based on the estimated concentration of protein in the medium.
    NOTE: Loading buffer should be stored at 4°C. Using β-mercaptoethanol and dithiothreitol (DTT) is not recommended due to their ability to destroy disulfide bonds between cysteine residues, which results in the inability of the MMP to refold after electrophoresis.
  2. Prepare the gel for the determination of total protein content by mixing samples in a 1:3 ratio with 4x reducing buffer [40% (v/v) glycerol, 240 mM Tris (pH 6.8), 8% (w/v) SDS, 0.04% (w/v) bromophenol blue, 5% (v/v) β-mercaptoethanol].
    NOTE: CAUTION! Wear gloves and goggles, and work under the fume hood. Loading buffer should be stored at -20 °C. The same amount of protein should be loaded into each lane. Samples can be optionally stored at -80 °C for a few days if required (STOP POINT 4).
  3. Incubate the samples for gelatin zymography for 20 min at 37 °C before running the electrophoresis.
    NOTE: Do not heat the samples at a temperature higher than 37 °C. Thermal denaturation of proteins may lead to the inactivation of protease activity or prevent the refolding of the enzymes causing false-negative results and wrong data interpretation31.
  4. Incubate the samples for total protein content determination for 10 min at 95 °C.
  5. Spin the samples for a few seconds in a small table centrifuge and load them into the wells of the gels.
  6. Optionally, load a molecular weight ladder and negative (samples that have been boiled for 10 min at 95 °C) and positive control samples (e.g., recombinant MMP-2 and MMP-9 or a cell line known to secrete MMP-2 and MMP-936) into the wells.
  7. Run electrophoresis, keeping it on ice until the dye flows out of the gel to provide good MMP-9 and MMP-2 band separation. Use the following SDS-PAGE electrophoresis conditions: initially, 20 mA per gel, increasing the power to 40 mA per gel when the samples enter the separating gel.
    ​NOTE: The maximum electric current should not exceed 130 mA for four gels. Otherwise, the gel will get overheated during electrophoresis. Electrophoresis can be performed in a box containing ice or in a cold room (4 °C), if available.

5. Activation, staining, and destaining of the polyacrylamide gel

  1. Gel washing and activation
    1. Remove the zymography gel from between the short and spacer plates and transfer it to a plastic container with washing buffer [50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2, 2.5% (v/v) Triton X-100]. Prepare washing buffer on the day of gelatin zymography.
      NOTE: As Triton X-100 is very viscous, pipet it according to the rules for pipetting viscous liquids to add the same volume of Triton X-100 each time the buffer is prepared37.
    2. Incubate the gel twice in the washing buffer, each time for 30 min with gentle agitation to remove SDS from the gel and replace it with Triton X-100.
    3. Transfer the gel to a plastic tank containing incubation buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2).
      NOTE: For long-term storage, keep the incubation buffer at 4 °C. Prepare fresh buffer if there are any signs of microbial contamination or precipitation.
    4. Incubate the gel in the incubation buffer for 12-20 h (here 16 h) at 37 °C.
      NOTE: The thickness of the gel and amount of loaded protein influence the incubation period in the incubation buffer, which should be optimized to obtain the areas of well-digested gelatin in the gel.
  2. Gel staining and destaining
    1. Prepare the staining solution containing an aqueous solution of 0.5% (w/v) Coomassie Brilliant Blue R-250, 30% (v/v) of ethanol, and 10% (v/v) of acetic acid.
      NOTE: CAUTION! Work under the fume hood and wear gloves when using acetic acid. Dissolve the weighed amount of Coomassie Blue in water, add ethanol, and mix the solution. In the end, slowly add acetic acid to the ethanol-water solution to avoid an exothermic reaction.
    2. Prepare the destaining solution containing 30% (v/v) of ethanol and 10% (v/v) of the acetic solution in H2O.
      NOTE: Mix ethanol with H2O. Add acetic acid slowly to the ethanol-water solution at the end to avoid an exothermic reaction.
    3. Transfer the gel to a plastic container with Coomassie Brilliant Blue solution and stain the gel for 30 min at room temperature with gentle agitation.
      NOTE: FastGene Q-Stain can also be used for staining the gel. The advantage of this dye is that it is ready to use and that the stained gel does not require any destaining step.
    4. Incubate the gel in the destaining buffer at room temperature with gentle agitation to destain. Continue the destaining until clear bands representing the gelatinase activity are visible.
      NOTE: If the staining with Coomassie Brilliant Blue solution is very intense, change the destaining buffer a few times during incubation.
    5. Keep the gel moisturized and do not allow it to dry out until its visualization.
      ​NOTE: Replace the destaining buffer with water to slow down the destaining process until visualization.

6. Visualization of proteins in polyacrylamide gels

NOTE: See the Table of Materials for details on the imaging system and software used to visualize proteins in polyacrylamide gels (Figure 2A-C). Other readily available gel visualization systems (such as iBright Imaging Systems, UVP PhotoDoc-It Imaging System, E-Gel Imager, and Azure Imagers) can also be used for this purpose.

  1. Switch imaging equipment on and open the software.
  2. Click File | New project to open a new project.
  3. Choose the application type by clicking Select... | Protein gels | Coomassie blue.
  4. Transfer the gels, one by one, into the imaging system and check the gel position using the Position Gel button.
  5. Visualize the gel by clicking Run protocol.
  6. Save the image as .scn to allow further analysis using the referenced software.
  7. Additionally, export file as .tif by clicking File | Export | Export for Analysis, which will be useful for analysis using ImageJ (STOP POINT 5).

7. Data analysis

  1. Gel analysis using the imaging software
    1. Open the imaging software and images of the zymogram and the gel representing the total protein content.
    2. Invert the image to obtain black bands on a white background. Under Go to the Image transform setting, click on the Invert image display.
    3. Select lines and bands on zymogram representing MMP-2 and MMP-9 manually or automatically using the Analysis Tool Box | Lane and Bands section. Create one band per line and adjust its size to include the whole line area for total protein content determination on the gel representing the total protein content.
    4. Adjust the lines and bands using the tools available in the Lane and Band section (Figure 2A'-C').
    5. Go to the Analysis Table and copy the Volume (intensity) and molecular weight of each lane and band (Figure 2A"-C").
    6. Divide the Volume of each lane per Volume of the selected control lane (here A375) to compare the total protein content between lanes (Figure 2A").
    7. Divide the Volume of each band per the value of the corresponding lane (from step 7.6) to normalize the Volume of MMP activity to the total protein content (Figure 2B",C").
    8. To compare the activity of gelatinases between lines, divide the normalized Volume of the studied line/studied condition per normalized Volume of the control line/standard condition (here A375) (Figure 2B",C").
  2. Alternative gel analysis using ImageJ software
    1. Open the ImageJ software and open the inverted gel image (black bands on a white background).
    2. Outline the first lane with gelatin-digested bands using the rectangle tool.
    3. Choose Analyze | Gels | Select First Lane in the ImageJ toolbar to select and mark the first lane.
    4. Transfer the first outlined selection (yellow rectangle) into the next lane and click Analyze | Gels | Select Next Lane to select and mark the next lane.
    5. Select all lanes presented on the gel in the same way (Figure 3A-C).
    6. Go to Select Analyze | Gels | Plot Lanes to make the lane profile plots (Figure 3A'-C').
    7. Using the Straight tool, separate bands with vertical lines at the edges of each peak and then draw horizontal lines to "close" their area (Figure 3A'-C'; yellow lines).
    8. Measure the size of each peak using the Tracking tool. Click in the area of each peak to select it and wait for the peaks to be outlined in yellow.
    9. Look for the size of each selected peak in the Results table (Figure 3A"-C").
    10. Normaize the data to the total protein content as described in step 7.1.
    11. Divide the "area" of the studied line/condition per "area" of the control line/standard condition (Figure 3A"-C").
  3. Data presentation
    1. Show the results obtained with the imaging software as the MMP activity normalized to total protein content and control line (Figure 4A,B).
    2. Present the results obtained with ImageJ as the MMP activity normalized to total protein content and control line (Figure 4C,D).

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Results

In this protocol, we describe the procedure for gelatin zymography (Figure 1) using media obtained from five melanoma cell lines (A375, SK-MEL-28, Hs-294T, WM9, WM1341D) as samples. The zymography approach shown here includes two separate SDS-PAGE electrophoreses. One SDS-PAGE electrophoresis results in the Coomassie Blue-stained gel, representing the total protein content loaded in each line (Figure 2A). It is used for the normalization of gelatinase activity d...

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Discussion

Despite the "step by step" protocol elaborated here, gelatin zymography requires optimization depending on the samples/cell lines being analyzed. Different cell types and cell lines (melanoma cell lines shown here) may secrete both forms (pro- and active) of MMP-2 and MMP-9 but with different gelatinase activity. The optimization of the procedure includes mainly the duration of cell starvation, the thickness of the polyacrylamide gel, the amount of loaded protein, and the duration of gel incubation in the incubat...

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Disclosures

The authors declare no competing financial interests.

Acknowledgements

This work was supported by the National Center for Science, Poland (project #2016/22/E/NZ3/00654, granted to AJM).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
22 µm syringe filtersNest331011
Acetic acid, 80% solutionChempur115687330
Acrylamide/bis-acrylamide, 30% Solution, 37.5:1BioshopACR010.500
Amicon Ultra-15 Centrifugal Filter UnitsMilliporeUFC901008ultracentrifugal filter units with 10 kDa cutoff
 Ammonium Persulfate (APS)Sigma-AldrichA-3678
Antibiotic-AntimycoticGibco15240062
Bradford reagentSigmaB6916
Bromophenol bluePolskie Odczynniki Chemiczne184070219
Calcium chloride dihydrate - CaCl2 · 2H2OSigma-AldrichC-5080
ChemiDoc SystemBio-radimaging system
Coomasie Brilliant Blue R-250Merck1.12553.0025
EthanolChempur1139641800
FastGene Q-StainNIPPON Genetics EUROPEFG-QS1
Fetal Bovine Serum - FBSGibco10270-106
Gelatin from porcine skinSigma-AldrichG-8150
GlycerolSigma-AldrichL-4909
glycineBioShopCAS #56-40-6
high glucose Dulbecco’s modified Eagle’s medium with reduced concentration (1.5 g/l) of NaHCO3Pracownia Chemii Ogólnej IITD PAN11-500
ImageJ software (Fiji)https://imagej.nih.gov/ij/version 1.52p
ImageLab softwareBio-rad
L-GlutamineGibco25030-024
Mini-PROTEAN Tetra CellBio-Rad#1658001EDU
N,N,N′,N′-Tetramethylethylenediamine (TEMED)Sigma-AldrichT9281
PageRuler Prestained Protein LadderThermo Fisher Scientific26616
Pierce BCA Protein Assay KitThermo Fisher Scientific23225
PowerPac Basic Power SupplyBio-rad1645050EDU
Sodium chloride - NaClChempur7647-14-5
Sodium dodecyl sulfate (SDS)Sigma-AldrichL4509
Tissue-culture 75 cm2 flaskVWR10062-872
Trisma baseSigma-AldrichT1503
Triton X-100Sigma-AldrichX100

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