RIPA lysis buffer was used to obtain protein samples. Next, 10% SDS-PAGE separated proteins. And protein samples were incubated in PVDF membranes with 5% non-fat milk. Next, protein samples were incubated overnight at 4 °C with RB1, E-cadherin, N-cadherin, Vimentin, PI3K, AKT, p-PI3K, p-AKT, and GAPDH primary antibodies (Abcam, Cambridge, MA, USA). Afterwards, goat polyclonal anti-rabbit IgG secondary antibodies (Abcam, USA) were added to incubate protein samples for 1 h. Finally, ECL (ECL, Pierce) was used to measure protein expression levels. And protein was quantified with the Image Lab Software (Bio-Rad, Kidlington, UK).
Gapdh primary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The GAPDH primary antibody is a tool used in research applications to detect the presence and quantity of the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein in biological samples. GAPDH is a widely used internal control and reference marker in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Abcam (5 mentions)
N cadherin, by Abcam (5 mentions)
Vimentin, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Bcl 2, by Abcam (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Ecl kit, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
P akt, by Abcam (1 mentions)
P pi3k, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
West pico chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Ecl solution, by Bio-Rad (1 mentions)
Goat polyclonal anti rabbit igg secondary antibody, by Abcam (1 mentions)
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Gst π, by Abcam (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Sds page, by Merck Group (1 mentions)
21 protocols using gapdh primary antibody
1
Western Blot Analysis of Cell Signaling
2
Protein Expression Analysis by Western Blot
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RIPA lysis buffer (Beyotime) was used to obtain protein samples. Next, 10% SDS-PAGE was used to separate protein. Protein samples were transferred to PVDF membranes. Blocked with 5% non-fat milk, protein samples were incubated overnight at 4 °C with E-cadherin, N-cadherin, Bcl-2, Bax and GAPDH primary antibodies (Abcam, Shanghai, China). Afterwards, secondary antibodies (Abcam, USA) were added to incubate protein samples for 1 h. ECL kit (Beyotime) was used to assess protein bands.
3
Western Blot Analysis of Protein Expression
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RIPA lysis buffer was used to obtain protein samples. Next, 10% SDS-PAGE was used to separate 25 μg of protein. Protein samples were transferred to the PVDF membrane. Blocked with 5% skimmed milk, the protein samples were incubated with vimentin, E-cadherin, N-cadherin, and GAPDH primary antibodies (Abcam, Cambridge, MA, USA) at 4°C overnight. Then, protein samples were incubated with HRP-conjugated secondary antibody (Abcam, USA) for 1 h. Finally, ECL (ECL, Pierce) was used to measure protein expression. Quantity One 4.52 analysis software was used to measure the gray value of the band. Relative expression of target protein (IOD) = gray value of the target protein/gray value of internal reference GAPDH.
4
Protein Expression Profiling in Cell Samples
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Protein samples were acquired by using RIPA lysis buffer (Beyotime). Protein was separated by 10% SDS-PAGE. Protein samples were transferred to PVDF membranes. Blocked with 5% non-fat milk, protein samples were incubated overnight at 4°C with E-cadherin, N-cadherin, vimentin, Bcl-2, Bax and GAPDH primary antibodies (Abcam). After washing, protein samples were incubated with secondary antibodies (Abcam) for 1 h. ECL kit (Beyotime) was used to assess protein bands. In addition, protein was quantified with Image Lab Software (Bio-Rad).
5
Quantitative Protein Expression Analysis
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Total cellular protein extracts were prepared on ice using a PRO-PREP protein extract solution (Intron, Seoul, Korea). Cell lysates were loaded onto a sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane for 1 h. Membranes were incubated 4°C overnight with cleaved PARP, active caspase-3, PTX3, or GAPDH primary antibodies (Abcam) diluted 1:1000 with 5% bovine serum albumin in Tris-buffered saline-Tween 20 (TBS-T). After incubation, the membranes were washed with TBS-T, and secondary antibodies (1:10000; horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG) were applied at room temperature for 1 h. Labeled bands were detected using a West Pico chemiluminescence kit (Thermo Scientific, Rockford, IL, USA).
6
Western Blot Analysis of Protein Expression
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The protein samples were extracted from mouse lung tissues and treated cells by lysis buffer, and the concentration was measured by BCA kit. Samples with equal amounts of protein were loaded into 10% SDS polyacrylamide gels and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with 1% BSA in TBST for 1h at room temperature and then incubated overnight at 4 °C with 1:1000 dilutions of p38, p-p38 and GAPDH primary antibodies (Abcam, Cambridge, UK) separately, followed by the incubation with 1:3000 dilutions of HRP-labeled secondary antibody for 30 min. After washing in TBST three times, the membranes were exposed to ECL solution (Bio-Rad, CA, USA) and imaged by chemiluminescence.
7
Protein Expression Analysis via Western Blot
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The protein samples were obtained using RIPA lysis buffer. Then the proteins were separated through a 10% SDS-PAGE and incubated with 5% non-fat milk in PVDF membranes at room temperature. Next we incubated the membranes overnight at 4℃ with E-cadherin, N-cadherin, vimentin and mTOR, GAPDH primary antibodies (1:1000; Abcam, USA). After washing, they were incubated with goat polyclonal antirabbit IgG secondary antibody (1:2000; Abcam, USA). Then, protein expression levels were measured by ECL (ECL, Pierce).
8
Western Blot Analysis of Protein Expression
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Protein samples were isolated by using RIPA lysis buffer (Beyotime Biotechnology). Then, the protein samples were separated by 10% SDS-PAGE. After transferring to the PVDF membrane, the protein was blocked with 5% skimmed milk. Next, the protein samples were incubated with Bax, Bcl-2, MRP1, P-gp, GST-π, and GAPDH primary antibodies (Abcam, Cambridge, MA, USA) at 4°C overnight. After washing 3 times, the protein samples were incubated with the corresponding secondary antibody (Abcam, USA) for 2 h. Finally, the ECL detection system (Thermo Fisher Scientific, Inc.) was used to visualize the blots. The relative level of protein expression was analyzed using ImagePro plus software (version 6.0; Media Cybernetics Inc., Rockville, MD, USA) and is represented as the density ratio versus GAPDH.
9
Quantifying Autophagy Proteins by Western Blot
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BCA (ABCAm) was utilized for protein quantification after RIPA lysis (ABCAm) of the cells. The proteins were then transferred to a PVDF (Sigma-Aldrich) membrane via SDS-PAGE (Sigma-Aldrich), sealed with 5 % skim milk for 2h, and added with LC3-II, Beclin-1 and GAPDH primary antibodies (1:1,000, ABCAm). After incubation at 4 ℃ overnight, an HRP labeled secondary antibody (1:5,000, ABCAm) was added to incubate at an ambient temperature for 1h, followed by development using ECL, and gray value analysis with Image J.
10
Western Blot Protein Expression Analysis
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CDK6 and β-actin protein expression was performed as previously described [14 (link)]. The primary antibody for TCEA3 was purchased from ProteinTech (ProteinTech Group, Wuhan, China). GAPDH primary antibody was purchased from Abcam (Abcam, USA).
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