Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines

Abstract

Epichaperomes are disease-associated pathologic scaffolds, composed of tightly bound chaperones, co-chaperones, and other factors. They mediate anomalous protein–protein interactions inside cells, which aberrantly affects the function of protein networks, and in turn, cellular phenotypes. Epichaperome study necessitates the implementation of methods that retain these protein complexes in their native cellular states for analysis. Here we describe a protocol for detection and composition analysis of epichaperomes in cell homogenates through native polyacrylamide gel electrophoresis.

1. Introduction

Polyacrylamide gel electrophoresis (PAGE) in the presence of a detergent-like sodium dodecyl sulfate (SDS) and presence or absence of denaturing and reducing substance is the most used technique for the separation of proteins. The introduction of SDS was the first hallmark in protein electrophoresis [1, 2]. SDS imparts a uniform negative charge to any protein (around 1.4 g/1 g of protein), making the individual charge of the protein irrelevant in terms of its mobility in a polyacrylamide gel [3]. SDS forms micelle around the protein molecules with a net negative charge depending on the size of the protein. Thus, the mobility of the protein is determined by the size of the protein effectively. Although SDS-PAGE is a very powerful technique for resolving a mixture of proteins in a sample, the obvious disadvantage is that the samples are boiled in Laemmli buffer that contains SDS and sometimes reducing agents like β-mercaptoethanol (β-ME) or dithiothreitol (DTT), which disintegrates most of the higher molecular weight complexes. However, electrophoresis can also be performed in the absence of any detergent. Under such conditions, protein complexes may be retained. The separation will be driven by charge and be dependent mainly on the primary sequence and structure of the protein and the pH of the electrophoresis buffer. This method of separation of proteins and complexes is referred to as native-PAGE.

Native-PAGE allows for the separation from cells and tissues of protein complexes in their native states. The method can be used for identification of physiological protein–protein interactions, oligomeric states, and native protein mass. These native complexes can be recovered from gels using diffusion and electroelution and can be subjected to downstream applications, like electron microscopy and 2D crystallization, or for further analysis for activity [4]. Thus, native-PAGE serves as an important tool for the analysis of higher molecular weight complexes isolated from tissues and cells to understand the changes in pattern and activity under different physiological conditions.

Here we demonstrate the use of native-PAGE for the study of epichaperomes. Not to be confused with folding chaperones, which have evolved to be dynamic and short-lived [5–7], epichaperomes are long-lived heterooligomeric assemblies of tightly bound chaperones, co-chaperones, and other factors [8–17]. Functionally, epichaperomes are also distinct from chaperones. They act as pathologic scaffolds remodeling protein–protein interaction networks, rather than serving as folders of proteins in protein synthesis and degradation pathways [8–17]. Epichaperomes are principally localized to diseased cells and tissues and represent a fraction of the total chaperone pools [8–17]. In contrast, the ubiquitous folding chaperones are abundantly expressed in all cells and across normal and disease conditions [18, 19]. Epichaperomes composition is context-dependent, meaning that a complement of epichaperome structures, each with distinct composition, forms in distinct disease conditions. For example, in cancer cells, heat shock protein 90 (HSP90) recruits heat shock cognate 70 (HSC70), HSP70-HSP90 organizing protein (HOP), and HSP110 and other co-chaperones and other factors, to function as a network to provide a survival advantage to cancer cells and tumor-supporting cells in the microenvironment [8, 14, 20]. In Parkinson’s disease, within midbrain dopaminergic neurons exposed to toxic stressors (e.g., rote-none), HSP90 recruits HSP60 into epichaperomes, and these epichaperome structures act to aberrantly rewire the interaction of proteins involved in dopamine synthesis pathways [10]. In neurons exposed to genetic stressors (e.g., PARKIN mutation), HSP90 recruits HSC70, HOP, HSP40, and several other co-chaperones to epichaperomes, to rewire the interaction of numerous proteins involved in inflammatory signaling pathways [10]. HSC70 is also an epichaperome constituent along with HSP90 in Alzheimer’s disease where epichaperomes negatively impact the interaction of proteins integral for synaptic plasticity [12]. Methods and protocols to study the presence and abundance of these disease-promoting heterooligomers in distinct biological contexts are of high importance.

Here, we describe in detail the step-by-step protocol for identification of HSP90-incorporating epichaperome complexes using native-PAGE from cell lines and evaluation of total chaperone levels using SDS-PAGE (Fig. 1). We discuss the impact of sample storage and handling on epichaperome stability (Fig. 2), the effect of gel gradient on the separation and detection of epichaperomes (Fig. 3), and the influence of gel type on epichaperome separation and detection efficiency (Fig. 4). Finally, we confirm the reproducibility of the protocol in the hands of a second investigator (Fig. 5).

Fig. 1.

Electrophoresis system setup. Snapshots of the setup at different stages, as described in the protocol, are provided. a: Cell lysates (100 μg) were loaded onto the gel. Pre-run, for gel equilibration, was done at 100V (cold room). The gel was run at 125V in the cold room. The run time for the 4 to 8 % hand cast gels was 2 h, for the 4 to 10% hand cast gel was 2.5 h, and for the 4 to 20% precast gel (Biorad) was 4 h. Transfer was done with 0.02% SDS in 1× transfer buffer for 2 h (cold room). PVDF membrane was used for the protein transfer process, unless otherwise indicated. b: Setup for Invitrogen 4 to 12% precast gels. The run time for the 4–12% precast gel (Invitrogen) was 3.5 h

Fig. 2.

The impact of sample storage and handling on epichaperome stability. Cell lysates (100 μg) were loaded onto the gel (Invitrogen precast 4–12% gradient gel) freshly prepared (1), or after being stored as indicated. For (2) lysates were stored at −20 °C but thawed at least 6 times before use. Pre-run, for gel equilibration, was done at 100V (cold room). The gel was run at 125V in the cold room for 3.5 h. Transfer was done with 0.02% SDS in 1× transfer buffer for 2 h (cold room). PVDF membrane was used for the protein transfer process

Fig. 3.

The effect of gel gradient on the separation and detection of epichaperomes in the MDA-MB-468 cancer cell line. Several chaperone and co-chaperone components of epichaperomes were detected as indicated. Setup same as in Fig. 1 for cell lysates (100 μg) loaded onto the Invitrogen precast 4–12% gradient gel or the Biorad 4 to 20% gradient gel

Fig. 4.

The influence of gel type on epichaperome separation and detection efficiency. a: Native PAGE profile of epichaperomes in the indicated cancer cell lines. CCD-18 contains little to no epichaperomes and is used as control to show the biochemical signature of chaperones on Native PAGE. Note: the protocol used is same as in Fig. 1, but a roller was used to remove air bubbles, which stretched the gel. Most affected are the 4–12 % precast Invitrogen gels (1mm thickness). The run time for the 4–8 % hand cast gels was 2 h, for the 4 to 10 % hand cast gel was 2.5 h, for the 4–12 % precast gel (Invitrogen) was 3.5 h, and for the 4 to 20% precast gel (Biorad) was 4 h. The hand cast gel was stored at 4 °C for one month prior to use. b: SDS PAGE profile (i.e., total levels) of chaperones in samples from panel (a). Note: additional bands for HSP110 and HSC70 are due to incomplete membrane stripping prior to re-blotting

Fig. 5.

Reproducibility by a second investigator. Same as in Fig. 4 for samples run by a different investigator using hand cast gels. A nitrocellulose membrane was used for transfer in this case (for the Native gel only)

2. Materials

Prepare all solutions using deionized water at 25 °C and analytical-grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing of waste materials. This protocol uses cancer cells. In order to use biologicals requiring biosafety level 2 (BSL2), the project was registered with the Institutional Biosafety Committee (IBC), and the personnel was trained in proper handling and use of hazardous materials. Biohazardous materials were handled and disposed of according to applicable state and federal regulations.

2.1. Cell Culture

1. Cell lines: (a) breast cancer cell lines, MDA-MB-468 (Catalog# HTB-132, RRID: CVCL_0419) and MDA-MB-453 (Catalog# HTB-131, RRID: CVCL_0418), (b); pancreatic cancer cell lines, ASPC1 (Catalog# CRL-1682, RRID: CVCL_0152) and MiaPaCa2 (Catalog# CRL-1420, RRID: CVCL_0428); and (c) normal colon fibroblasts, CCD-18 (Catalog# CRL-1459, RRID: CVCL_2379) were purchased from American Type Culture Collection (ATCC). Vials are stored in liquid N₂ when not in use (see Note 1).

2. Gibco^™ Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with L-glutamine, from Fisher Scientific (Catalog #11–965-084), for culturing of cells (stored at 4 °C) (see Note 2).

3. Gibco^™ Fetal Bovine Serum (FBS), from Fisher Scientific (Catalog #10-082-147), for culturing of cells (stored at −20 °C, in 50 mL aliquots, thawed on ice prior to use).

4. Corning^™ Penicillin-Streptomycin Solution (100x), from Fisher Scientific (Catalog #MT30002CI, storage temperature − 20 °C).

5. Gibco^™ PBS (pH 7.4), from Fisher Scientific (Catalog #10-010-023). Store at 4 °C, brought to room temperature (RT) before use.

6. Gibco^™ Trypsin-EDTA (0.25%), from Fisher Scientific (Catalog #25-200-056), for cell trypsinization (storage temperature, −20 °C, thawed before use).

7. 100 mm cell culture dish from Fisher Scientific (Catalog #08-772-6). Store at RT.

8. 175 cm² cell culture flask from Fisher Scientific (Catalog #10-126-13). Store at RT.

9. CO₂ incubator from NuAire (Catalog #NU-8700).

2.2. Cell Lysate Preparation for Native-PAGE

• 1Gibco^™ PBS (pH 7.4), from Fisher Scientific (Catalog #10-010-023). Store at 4 °C.
• 2Cell scraper, from Fisher Scientific (Catalog #080-100-240). Store at RT.
• 3Lysis buffer (1 x native lysis buffer, 20 mM Tris pH 7.4, 20 mM KCl, 5 mM MgCl₂, 0.01% NP40): Mix 10 mL of 1 M Tris-HCl (pH 7.4), 10 mL of 1 M KCl, 2.5 mL of 1 M MgCl₂, and 0.5 mL of 10% NP40. Make up to 500 mL with deionized water. Store at 4 °C. Take 10 mL of this solution and add one Complete^™ Protease inhibitor cocktail tablet (Catalog #11697498001) and one PhosSTOP^™ phosphatase inhibitor cocktail tablet (Catalog #4906837001) to make native lysis buffer for cell lysis (stored at 4 °C and taken out on ice before use).
• 4Dry ice.
• 5Pierce^™ BCA protein assay kit from Thermo Scientific (Catalog #23225) for protein estimation. Store at RT.
• 6SpectraMax Paradigm Multi-Mode Microplate Reader from Molecular Devices (or other plate readers with absorbance measurement capabilities).
• 7Centrifuge from Eppendorf (Catalog #5810R).
• 8Benchtop centrifuge from Eppendorf (Catalog #5417R).
• 9Fisher Scientific Isotemp 220 Digital Water Bath (Product Code: FS-220).

2.3. Native Polyacrylamide Gel

• 10Running gel buffer (1 × Tris-glycine native buffer, 25 mM Tris, 192 mM glycine, 0.1% SDS): Dissolve 3.0 g of Tris-base and 14.4 g of glycine in 500 mL of deionized water, and make up to 1000 mL with deionized water. Store at 4 °C (see Note 3).
• 11Acrylamide/bis-acrylamide 37.5:1 (40% solution) from Fisher Scientific (Catalog #BP1410-01). Store at 4 °C.
• 121 M Tris-HCl (pH 8.0) form TEKnova (Catalog #T1080). Store at room temperature.
• 13Transfer buffer (25 mM Tris pH 8.3, 192 mM glycine, 20% methanol): Dissolve 3.0 g of Tris-base and 14.4 g of Glycine in 500 mL of deionized water, and make up to 800 mL with deionized water. Add 200 mL of methanol to make the final volume of 1000 mL. Add 2 mL of 10% SDS solution per 1000 mL of the transfer buffer before the protein transfer procedure. Store at 4 °C.
• 14Ammonium persulfate solution (10%): Dissolve 1 g of ammonium persulfate in 5 mL of deionized buffer, and make up to 10 mL with deionized water. Store at 4 °C. It is an oxidizing agent that is used with TEMED to catalyze the polymerization of acrylamide and bis-acrylamide.
• 15TEMED (N,N,N′,N′-tetramethylethylenediamine) from Fisher Scientific (Code# BP150-20). It is a free radical stabilizer and an essential catalyst for polyacrylamide gel polymerization. Store at 4 °C.
• 16Loading buffer (6×): Mix 3.75 mL of Tris-HCl 1 M, pH 8, 6 mL glycerol, and 6 mg bromophenol blue, and make up to 10 mL with deionized water. Preferable storage at −20 °C but can also be stored at 4 °C, if in regular use.
• 17Precast gels: NuPAGE^™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, mini protein gels from Thermo Fisher (Catalog #NP0321BOX), and 4 to 20% Mini-PROTEAN^® TGX^™ Precast Protein Gels, 10-well, 50 μL from BioRad (Catalog #4561094).
• 18Electrophoretic running unit: Mini-PROTEAN Tetra Vertical Electrophoresis Cell, 4-gel (Catalog #1658004EDU).
• 19Electrophoretic transfer unit: Mini Trans-Blot Electrophoretic Transfer Cell (Catalog# 1703930).
• 20Invitrogen Mini Gel Tank (Catalog #A25977) (suitable for the Invitrogen precast gels only).
• 21Gradient Maker from C.B.S. Scientific Co. (Model #GM-40) for the hand cast gels.
• 22Bio-Rad PowerPac Universal Power Supply (Catalog #1645070).
• 23Membrane for protein transfer: Immobilon^®-P PVDF (PolyVinyliDeneFluoride). Membrane (Catalog #IPVH00010) and Amersham Protran 0.2 μm Nitrocellulose Blotting Membrane (Catalog #10600006). Store at RT.
• 24Tris buffer saline Tween 20 (TBST): Mix 20 mL of 1 M Tris-HCl (pH 7.4) and 8.78 g of NaCl in 100 mL of deionized water. Make up to 1000 mL of deionized water, and add 1 mL of Tween 20. Store at RT.
• 25Blocking solution: Dissolve 0.5 g of bovine serum albumin (BSA) in 5 mL of TBST, and make up to 10 mL with TBST.
• 26Molecular weight marker: NativeMark^™ Unstained Protein Standard (Catalog #LC0725). Store at−20 °C.
• 27Ponceau S solution from MilliporeSigma (Catalog #P7170). Store at RT.
• 28Thermo Scientific SuperSignal^™ (SuperSignal West Dura Extended Duration Substrate is a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate) detection kit. Store at 4 °C.

2.4. Membrane Stripping

1. Stripping solution: 5 mM EDTA in deionized water. It is a solution for removing primary and secondary antibodies from probed Western blot membranes. Alternatively, a commercially made stripping solution can also be used.

2.5. SDS-Page

1. ProtoGel Resolving Buffer (4×) from National Diagnostics (Order# EC-892).

2. ProtoGel Stacking Buffer (4×) from National Diagnostics (Order# EC-893).

3. SDS-PAGE 1× running buffer: Dilute 100 mL of 10× TGS Buffer from Fisher Scientific (Catalog #BP1341) with deionized water to make the final volume up to 1000 mL.

4. Laemmli Sample Buffer (5×): Mix 1 g of SDS, 5 mL of glycerol, 3.125 mL of Tris-HCl (pH 6.8), 5 mg of bromophenol blue, and 0.775 g of DTT, and make up to 10 mL with deionized water.

5. Acrylamide/bis-acrylamide 37.5:1 (40% solution), ammonium persulfate solution, TEMED, TBST, and Ponceau S solution were prepared or purchased as mentioned above.

6. Transfer buffer (25 mM Tris pH 8.3, 192 mM glycine, 20% methanol): Dissolve 3.0 g of Tris-base and 14.4 g of glycine in 500 mL of deionized water, and make up to 800 mL with deionized water. Add 200 mL of methanol to make the final volume of 1000 mL.

7. Precision Plus Protein^™ Kaleidoscope^™ Prestained Protein Standards from Bio-Rad (Catalog #1610375). Store at −20 °C.

2.6. Detection

1. A panel of anti-chaperone antibodies has been screened to identify the ones recognizing the target protein in its native form. These native-cognate antibodies were used in the native-PAGE analysis of epichaperome assemblies: HSP90β (SMC-107, RRID:AB_854214, store at −20 °C) and HSP110 (SPC-195, RRID:AB_2119373, store at −20 °C) antibodies from StressMarq; HSC70 (SPA-815, RRID:AB_10617277, store at −20 °C) and HOP (SRA-1500, RRID:AB_10618972, store at −20 °C) from Enzo; and HSP90± (ab2928, RRID:AB_303423, store at −20 °C) from Abcam. All the above antibodies were used at 1: 2000 dilution. Anti-β-actin antibody (A1978, RRID: AB_476692, store at −20 °C) was purchased from Sigma-Aldrich and used at 1:5000 dilution.

2. Species-specific HRP-conjugated secondary antibodies were purchased from Southern Biotech (stored at 4 °C), and used at a 1:5000 dilution: anti-mouse (Catalog #1030-05), anti-rat (Catalog #3030–05), and anti-rabbit (Catalog #4010-05).

3. ChemiDoc MP imaging system from Bio-Rad (Catalog #17001402).

3. Methods

All procedures are performed at room temperature unless otherwise specified.

3.1. Cell Culture and Cell Lysate Preparation for Native- and SDS-PAGE

1. Perform the cell culture in sterile conditions by operating under a Class II Biological Safety Cabinet.

2. Maintain the cells in culture at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1× penicillin and streptomycin and 10% FBS (Sigma) inside an incubator with 5% CO₂ supply (see Note 4).

3. For the experiment, seed the cells in 10 cm dishes (in 12 mL medium) and grow till 80% confluency to harvest for cell lysis (see Note 5).

4. Discard the media and wash the cells with ice-cold 1 × PBS, and then scrape off using cell scrapers. Cell scrapers should be washed with ice-cold 1× PBS before use. Collect the cell suspension in 1 × PBS following by centrifugation at 3500 rpm for 5 min at 4 °C. Discard the supernatant and add the native lysis buffer at around 1.5 times the cell pellet volume.

5. For lysis, incubate the samples on dry ice for 10 min. Remove the sample from dry ice, and incubate it at 37 °C water bath for 2 min. Repeat the procedure three times followed by incubation on ice for 30 min. Centrifuge the lysates at 12,000 rpm for 20 min at 4 °C.

6. Collect the supernatant and determine the protein concentration with the BCA kit.

7. Prepare the samples for loading onto the native gel by mixing 100 μg of lysate with 6× loading dye (see Note 6).

3.2. Cell Lysate Preparation for SDS-PAGE

1. For sample preparation, heat 20 μg of protein in 5× Laemmli buffer at 95 °C for 10 min.

3.3. Preparation of Precast Gels for Running Native-PAGE

1. Take the NuPAGE^™ 4 to 12% gel out of the plastic bag. Remove the white tape at the bottom of the gel. Place the gel into the Invitrogen mini gel tank, and take out the comb. Clear out the individual lanes using a 1 mL syringe and fill the tank with 1× cold native running buffer (approximate volume 500 mL, maintained at 4 °C). If using the 4 to 20% Mini-PROTEAN^® TGX^™ Precast Protein Gel, place the gel in the Mini-PROTEAN Tetra Vertical Electrophoresis Cell, and take out the comb. Clear the lines using a 1 mL syringe and fill the tank with 1× cold native running buffer (i.e., maintained at 4 °C).

2. Pre-run the gel without the samples for 1 h at 100 V in the cold room (i.e., maintained at 4 °C).

3.4. Preparation of Handmade Continuous Gels for Running Native-PAGE

1. Clean and dry the glass plates by whiping them with a tissue paper. Thoroughly clean the 1.5 mm spacers and the comb. Assemble the glass plates, spacers, and comb as described by the manufacturer.

2. Prepare the high- and low-percentage gel solutions as per Table 1. (see Note 7).

3. Prepare the gradient generator (see Note 8). Ensure that the generator is clean and without bubbles inside the tube and in the channel connecting both chambers.

4. A gradient generator consists of two chambers, A and B, connected to each other. Chamber B contains the low-percentage non-denaturing polyacrylamide gel (4%) and chamber A contains the high-percentage non-denaturing polyacrylamide gel (8%, 10%). A plastic tubing, through which the gel gets poured into the gel cassette, is connected to the lower bottom of container A.

5. After pouring the gels into the appropriate chambers, allow approximately 0.3 mL of the gel solution to flow through the tubing to ensure continuous gel flow and removal of air bubbles. Then, open the valve between the chambers allowing for the low-percentage gel to mix with the high-percentage gel. Make sure the flow rate is low. A faster flow will cause turbulence in the gel and may disrupt the gradient.

6. Attach a 200 μL pipette tip to the free end of the tube, and insert it into the assembled glass plates to allow the gradient gel to be poured in. Fill up the cassette with the gel.

7. After the process is complete, wash the gradient generator unit immediately to avoid acrylamide polymerization inside the gradient generator.

8. Place the comb into the gel cassette.

9. Set the gel aside for 20–30 min for the gel to polymerize.

10. Load the samples into the native gel wells. Use one well to add the molecular weight marker (8 μL of unstained protein standard (NativeMark^™, Invitrogen)). Start the run at 125 V. Perform this step while in the cold room (i.e., maintained at 4 °C).

11. For 4% to 8% gradient gel, the run is continued until the dye front reaches the end of the gel, which takes approximately 2 h. For 4% to 10% gradient gel, the electrophoresis is continued to allow the dye front to run out of the gel, which takes approximately 2.5 h. For the 4% to 12% Invitrogen precast gel, the dye front is allowed to run out, which takes approximately 3.5 h. For the 4% to 20% Bio-Rad precast gel, the dye front is allowed to run out (approximately 4 h) (see Note 9).

Table 1.

Preparation of the hand cast gels for Native-PAGE

Components	4%a	8% a	10% a
40% Acrylamide (in mL)	1	1	1.25
Deionized water (in mL)	5.2	2.1	1.875
1 M Tris-HCl, pH 8 (in mL)	3.8	1.9	1.875
10% APS (in μL)	0.080	0.040	0.040
TEMED (in μL)	0.006	0.003	0.003

^aPolyacrylamide percentage

3.5. SDS-Page

1. Use the 4 to 20% precast gel from Bio-Rad or the 4 to 12% precast gel from Invitrogen for running the SDS-PAGE. Alternatively, prepare a hand cast gel (Table 2).

2. Clean and dry the glass plates, clean the 1.5 mm spacers and the comb thoroughly, and assemble the unit as described by the manufacturer.

3. Using a glass marker, draw a line on the glass plate around 3 cm below the top of the plate. This indicates the level to which the resolving gel will be poured.

4. Prepare both the stacking and the resolving gel solutions at room temperature by combining all reagents according to Table 2, except for APS and TEMED (see Note 10).

5. Add APS and TEMED to the resolving gel solution (see Table 2 for the specified amount), and mix well by swirling gently. Pour the acrylamide gel mixture into the gel cassette.

6. Overlay the gel with isopropanol.

7. Allow the gel to polymerize for 1 h.

8. Remove the water/isopropanol overlay from the top of the gel, wash with water, and drain the excess water with strips of filter paper.

9. Add APS and TEMED to the stacking gel solution, and then pour the stacking solution gently on top of the resolving gel. This should be done gently to avoid bubbles.

10. Place the comb carefully onto the top of the gel and allow the gel to polymerize.

11. Following polymerization, set the gel in the Bio-Rad apparatus and fill up the tank with the running buffer till the recommended mark.

12. Remove the comb carefully, clear the lanes using a 1 mL syringe, and then load 20 μg of sample into each well. Use precision Plus Protein^™ Kaleidoscope^™ Prestained Protein Standard as a molecular size marker.

13. Run the gel at 100 V at room temperature till the dye runs out of the gel.

Table 2.

Preparation of the hand cast gels for SDS-PAGE

Components	4% stacking	10% resolving
Acrylamide/bis-acrylamide 40%	0.8	5.32
Resolving gel buffer (mL)	–	5.32
Stacking gel buffer (mL)	2
Water (mL)	5.1	10.4
SDS 20% (μL)	40	106.4
APS10% (μL)	40	106.4
TEMED (μL)	8	8

3.6. Protein Transfer and Immunoblotting

1. After the electrophoresis run is over, disassemble the gel unit, and set up the transfer process. For the Invitrogen and Bio-Rad precast gels, carefully remove the plastic casing such that the gel is not torn apart.

2. Activate the PVDF membrane for transfer by incubating it in methanol for 2 min, followed by washing it in distilled water and 1 × cold transfer buffer.

3. Set up the gel for transfer in a way that the gel is on the cathode side (black side of the transfer cassette) and the PVDF membrane is placed on top of the gel facing the anode side (the transparent side of the cassette) (see Note 11).

4. Use a roller to remove any air bubbles between the gel and PVDF membrane (see Note 12).

5. Place the cassette into the transfer unit, fill it with 1× cold transfer buffer (kept at 4 °C), and run at 100 V for 2 h in a cold room (4 °C).

6. After the transfer, stain the membrane with Ponceau S solution to visualize the molecular weight marker for the native gel.

7. Block the PVDF membrane with the blocking solution for 1 h at room temperature.

8. Add primary antibodies, and incubate overnight (for 12 h to 16 h) in the cold room at 4 °C.

9. Wash the blots with TBST three times (10 min each) at room temperature.

10. Incubate the membrane with the corresponding secondary antibody for 2 h at room temperature.

11. Wash with 1× TBST three times and proceed to signal visualization.

3.7. Chemiluminescent Detection

1. Develop the blots using the Thermo Scientific SuperSignal^™ detection kit, which uses a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate, in a ChemiDoc MP system (Bio-Rad). Prepare the detection solution by mixing both components of the kit in a 1:1 ratio. Make approximately 0.1 mL detection solution for a 1 cm² membrane.

2. Take the membrane out of the TBST solution with tweezers, and remove excess buffer by holding the membrane in a vertical position, with the lower edge of the membrane touching a sheet of blotting paper.

3. Place the membrane on the developing tray.

4. Distribute the substrate evenly on the top of the membrane, incubate for 1 min, remove the solution, and insert the tray into the ChemiDoc MP machine for signal detection.

5. For re-blotting with a different antibody, the PVDF membranes can be stripped by boiling blots for 3 min in 5 mM EDTA in distilled water or by using commercially available stripping solutions.