Cells were harvested and lysed with 500 μL of lysis buffer (150 nmol/L NaCl, 50 nmol/L Tris-HCl (pH 7.4), 1% Triton X-100, 1 mmol/L EDTA (pH 8.0), and 0.1% sodium dodecyl sulfate (SDS)) with a protease inhibitor cocktail and then incubated on ice for 30 min. The supernatants were collected by centrifugation at 4 °C at 12,000 ×g for 30 min and then boiled in 5× SDS-PAGE loading buffer for 10 min. The prepared samples were resolved on 10% SDS-PAGE and then transferred onto nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% skim milk powder in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST) for 30 min before being incubated with primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Thermo Fisher Scientific) for 1 h. The primary antibodies were used as follows: mouse anti-Myc (1:10,000, MBL), mouse anti-HA (1:10,000, MBL), mouse anti-Flag (1:10,000, Sigma), rabbit anti-HA (1:10,000, Sigma), rabbit anti-Flag (1:10,000, CST), rabbit anti-Myc (1:10,000, Sigma), rabbit anti-VP30 pSer29 antibody (1:1000, ABclonal), rabbit anti-Na/K ATPase (1:1000, ABclonal), and mouse anti-GAPDH (1:1000, ABclonal). The secondary antibodies, goat anti-rabbit IgG and goat anti-mouse IgG, were used at a 1:5,000 dilution.
Mouse anti gapdh
Manufactured by ABclonal
Sourced in United Kingdom, United States, Australia
Mouse anti-GAPDH is a primary antibody that recognizes the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein. GAPDH is a widely used internal control and housekeeping gene in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti flag, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Rabbit anti ha, by Merck Group (1 mentions)
Rabbit anti myc, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Rabbit anti wt1, by Cell Signaling Technology (1 mentions)
Blocking solution, by Beyotime (1 mentions)
Ecl luminescent solution, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Rabbit anti pcna, by Abcam (1 mentions)
Ecl western blot kit, by Biosharp (1 mentions)
Chemidoc analysis system, by Bio-Rad (1 mentions)
Rabbit anti cdk2, by Abcam (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Bicinchoninic acid assay, by Beyotime (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Rabbit anti histone h3, by Abcam (1 mentions)
7 protocols using mouse anti gapdh
1
Protein Immunoblotting and Detection
2
Western Blot Analysis of Protein Expression
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After various treatments, total protein was extracted from the cells. Briefly, cells were lysed in an ice-cold extraction buffer (RIPA extraction buffer, 1 mM PMSF and 1× protease inhibitor cocktail). The concentration was monitored by the BCA method. Proteins were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Life Sciences, Waltham, MA, USA). Membranes were incubated with antibodies including primary rabbit anti-AKT1 (1:1000) (Abclonal, China), rabbit anti-phospho-AKT1 (1:1000) (Abclonal, China), mouse anti-phospho-γ-H2AX (1:1000) (Abcam, UK), mouse anti-GAPDH (1:1000) (Abclonal, Wuhan, China) at 4 °C overnight, followed by the addition of horseradish peroxidase-linked anti-mouse/rabbit IgG secondary antibodies (1:10,000) (Thermo) and ECL visualization of the bands.
3
Western Blotting of WT1 and Housekeeping Proteins
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Western blotting was performed as previously described with minor modifications (Meng et al., 2019a (link)). Briefly, the cells were washed twice with pre-cooled PBS, and RIPA buffer containing protease inhibitors was added to cells, which were lysed on ice for 20 min. The protein lysate was centrifuged at 4°C at 10,000 rpm for 5 min, and the supernatant was added to 5×SDS loading buffer at 100°C for 10 min. The denatured proteins were subjected to SDS-PAGE electrophoresis and were transferred to PVDF membranes (Millipore). After blocking with a blocking solution (Beyotime) for 20 min, the membrane was incubated in the primary antibody at 4°C overnight. The primary antibodies used were rabbit anti-WT1 (1:1,000, Cell Signaling Technology, Inc.), mouse anti-GAPDH, and mouse anti-ACTB (1:1,000, Abclonal). After washing, the Abclonal secondary antibody was incubated at 1:1,000 for 1 h. Finally, the ECL Luminescent Solution (Millipore) was used to visualize blots and for imaging of protein bands.
4
Western Blot Analysis of ATF4, SHH, and Gli1
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GC tissues or entire cells were used to extract equivalent amounts of protein, which were then separated with 10% SDS-PAGE gel and electro-transferred onto polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). The antibodies were incubated with various primary antibodies overnight at 4 °C after blocking in 5% milk for 2 h; rabbit anti-ATF4-1 (1:3000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-SHH (1:3000, Cell Signaling Technology, Danvers, MA, USA), mouse anti-Gli1 (1:3000 Santa Cruz Bicycles, California, CA, USA), rabbit anti-GAPDH (1:5000, Cell Signaling Technology, Danvers, MA, USA), and mouse-anti GAPDH (1:5000, from Abclonal, Wuhan, China) antibodies were used as internal controls. Signals were then detected using an upgraded chemiluminescence substrate after incubating with a secondary antibody bound to peroxidase (Millipore, Schwalbach, Germany). Image J was used to measure the intensity of the bands on the Western blot.
5
Protein Expression Analysis of DPCs
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After 48 h of transfection, the DPCs proteins were disposed of with RIPA lysis buffer (Beyotime, Shanghai, China), and concentrations were detected using the BCA method. The proteins were separated and then transferred to PVDF membranes, which were probed with 1:500 rabbit anti-EGR1 (Affinity, Melbourne, Australia), 1:2500 mouse anti-GAPDH (ABclonal, Wuhan, China), 1:1000 rabbit anti-PCNA (Abcam, Cambridge, UK), 1:1000 rabbit anti-CDK2 (Abcam, Cambridge, UK), 1:3000 goat anti-rabbit IgG HRG antibody (ABclon, Wuhan, China), and 1:3000 goat anti-mouse (ABclonal, Wuhan, China). The protein expressions were measured using the ECL Western Blot kit (BioSharp, Hefei, China), and analysed by the ChemiDocTM Analysis System (Bio-Rad, Hercules, CA, USA).
6
Protein Extraction and Western Blotting of Mouse Testicular Tissue
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Mouse testicular tissue was lysed using nuclear and cytoplasmic protein extraction kit (Beyotime, P0027) according to the manufacturer’s instructions. The lysates were ultracentrifuged at 12000 g for 10 min at 4 °C. The supernatants and remaining sediment were collected separately. The concentration of protein in the sediment and supernatants were measured using a bicin-choninic acid assay (Beyotime Biotechnology, P0012S). For western blot assay, 50 μg of protein samples was loaded on 10% SDS-PAGE gel and runed 1.5 h at 100 V before transferring to PVDF membranes. The antibodies used were as follows. Rabbit anti-CDKN2AIP (1:1000, Proteintech, Cat No.16615-1-AP), Mouse anti-SYCP3 (1:1000, Abcam, Cat No. ab97672), Rabbit anti-histone H3(1:1000, Abcam, Cat No.ab1791), Rabbit anti-PRM1(1:1000, Affinity, Cat No.DF5045), Rabbit anti PRM2(1:1000, Proteintech, Cat No.14500-1-AP), Rabbit anti-SUN1(1:1000, Abcam, Cat No.ab103021), Mouse anti-GAPDH (1:10,000, ABclonal, Cat No.AC002), Goat Anti-Rabbit IgG H&L (HRP) (1:8000, Abcam, ab6721), Rabbit Anti-Mouse IgG H&L (HRP) (1:8000, Abcam, ab6728).
7
Cell and Tissue Protein Lysis Protocol
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