Based on the methods described in previous studies 8 (link), 9 (link), total proteins from the cells in each group were subjected to 12% SDS PAGE denaturing gel electrophoresis and transferred to PVDF membranes (Millipore) after the completion of electrophoresis. After blocking and membrane washing, primary antibodies (rabbit polyclonal antibody against human CCND1 (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal antibody against human CDK4 (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal antibody against human Neuritin (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal antibody against human GAPDH (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA)) were added, followed by incubation at 37 °C for 45 min. Secondary antibodies (peroxidase-linked goat anti-rabbit IgG (1 : 1000; Santa Cruz)) were added after sufficiently washing the membrane, followed by incubation at 37 °C for 45 min. The membrane was washed 4 times with TBST (Tris-buffered saline and Tween 20) at room temperature with 14 minutes per wash. The proteins in the membrane were detected with ECL enhanced chemiluminescence (ECL kit, Pierce Biotechnology). Densitomertic analysis was performed by Quantity One Software (Bio-Rad, Hercules, CA, USA).
Rabbit polyclonal antibody against human gapdh
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit polyclonal antibody against human GAPDH is a laboratory reagent used for the detection and quantification of the GAPDH protein in biological samples. GAPDH is a widely expressed enzyme involved in glycolysis and is commonly used as a housekeeping gene for normalization in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Peroxidase linked goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Fluorchem hd2 system, by Bio-Techne (1 mentions)
Ripa lysis buffer, by BestBio (1 mentions)
Goat anti mouse or anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by MultiSciences Biotech (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by BestBio (1 mentions)
Gi254023x, by Merck Group (1 mentions)
Human ccl2, by Thermo Fisher Scientific (1 mentions)
4 protocols using rabbit polyclonal antibody against human gapdh
1
Western Blot Analysis of Protein Expression
2
Quantification of CTLA-4 Expression in CIK Cells
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For protein analysis, total protein was extracted from 1×107 CIK cells using RIPA lysis buffer (BestBio, Shanghai, China) and then quantified using a BCA kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amount of whole cell lysates (20 µg per lane) were separated using SDS-PAGE on a 10% gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked using 2.5% non-fat milk for 1 h at room temperature and then incubated with rabbit monoclonal antibody against human CTLA-4 (cat. no. SC-9094; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or rabbit polyclonal antibody against human GAPDH (cat. no. SC-25778; 1:1,000; Santa Cruz Biotechnology, Inc.) for 2 h at 25°C followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (cat. no. SC-2004; 1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h at 25°C prior to detection with chemiluminescence (FluorChem™ HD2 system; ProteinSimple; Bio-Techne, Minneapolis, MN, USA). The expression of CTLA-4 was normalized to that of GAPDH.
3
Western Blot Analysis of Lung Cancer Cells
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Lung cancer cell lysates were prepared using RIPA buffer (BestBio, Shanghai, China) and equalized for protein concentrations with a BCA Kit (Pierce, Rockford, IL) according to the manufacturers' recommendations. An equal amount of whole cell lysates were resolved using SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) followed by incubation with primary mouse monoclonal antibodies against human EZH2 (BD Biosciences), flag (Multisciences, Hangzhou, China), rabbit polyclonal antibody against human GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal antibody against human NOTCH1 (Abcam, Shanghai, China) and goat anti-mouse or anti-rabbit IgG-HRP (Santa Cruz Biotechnology). The immunoreactive proteins were detected with SuperSignal® West Pico Chemiluminescent Substrate (Thermo, Rockford, IL) according to the manufacturer's instructions.
4
Characterization of ADAM10 in Cells
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R&D System (Wiesbaden, Germany): Mouse monoclonal antibody against human ADAM10 ectodomain (clone 163003); mouse monoclonal IgG2B isotype control (Clone 20116); goat polyclonal anti-Mouse IgG (H + L) secondary antibody (Alexa Fluor® 647). Mouse monoclonal antibody against human CD9 (MM2/57); Rabbit polyclonal antibody against human ADAM10 C-terminus was obtained from Invitrogen (Frankfurt, Germany), rabbit polyclonal antibody against human GAPDH from Santa Cruz Biotech (Dallas, TX, USA). Rabbit polyclonal antibody against beta actin from Abcam (Cambridge, UK). Mouse monoclonal antibody against flotillin-1 (Clon 18); mouse monoclonal antibody against human E-cadherin C-terminus from BD Biosciences (Heidelberg, Germany). The small molecule inhibitor for ADAM10 GI254023X was from Merck Millipore (Darmstadt, Germany), and human CCL2 was from Peprotech (Rocky Hill, NJ, USA).
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