Protein was extracted with complete RIPA buffer, and details about Western blot analysis can be found in our previously studies (16 (link)). Briefly, protein samples are boiled and separated on a 10% polyacrylamide gel and transferred to PVDF membranes (Invitrogen, Carlsbad, CA, USA), blocked with 3% BSA for 2 hours and incubated overnight with primary antibody (MET monoclonal antibody, 1:200 dilution; GAPDH monoclonal antibody, 1:200 dilution) (Santa Cruz Biotechnology). After being washed with PBST, immunoreactive bands were visualized through HRP-conjugated m-IgGk secondary antibody, 1:1,000 dilution (Santa Cruz Biotechnology) and ImageQuant LAS 350 (GE Healthcare).
Gapdh monoclonal antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The GAPDH monoclonal antibody is a laboratory tool used for the detection and quantification of the GAPDH protein. GAPDH, or glyceraldehyde-3-phosphate dehydrogenase, is a housekeeping gene that is commonly used as a reference protein in various experimental techniques. The GAPDH monoclonal antibody can be used in applications such as Western blotting, immunohistochemistry, and immunoprecipitation to identify and measure the GAPDH protein levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Imagequant las 350, by GE Healthcare (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mouse anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Sc 722, by Santa Cruz Biotechnology (1 mentions)
Anti hmmr, by OriGene (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Rabbit anti human caspase 3, by Abcam (1 mentions)
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mitochondria isolation kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Bax polyclonal antibody, by Cell Signaling Technology (1 mentions)
Bcl 2 monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Trans blot system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
11 protocols using gapdh monoclonal antibody
1
Western Blot Analysis of MET Protein
2
Investigating Glyoxalase-1 and Nrf2 Signaling
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RPMI-1640 medium and RIPA buffer were purchased from ThermoFisher Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS), trypsin, and penicillin-streptomycin were purchased from HyClone (Logan, UT, USA). The primary antibodies were anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Glo-1 monoclonal antibody (sc-133214; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-nuclear factor erythroid 2-related factor 2 (Nrf-2) monoclonal antibody (sc-722; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were mouse anti-rabbit IgG-HRP (sc-2357; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse IgG-HRP (AP124P; Sigma-Aldrich, St. Louis, MO, USA). JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine chloride was purchased from Biotium (Fremont, CA, USA). MG, N-acetyl-cysteine (NAC), and all other chemicals were purchased from Sigma-Aldrich.
3
Protein Extraction and Western Blot
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Protein extraction and Western blot analysis were performed as described [58 (link)] with following antibodies: rabbit polyclonal anti HMMR (1:100; Origene, Rockville, USA) and GAPDH monoclonal antibody (1:1000; Santa Cruz, Dallas, TX, USA).
4
Western Blot Analysis of TNF-α and Caspase-3
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Example 3
After transfection with various formulations, cells were lysed in 50 μl radioimmunoprecipitation assay (RIPA) buffer and were re-suspended in 50 μl 2× sodium dodecyl sulfate (SDS) sample buffer with 1% β-mercaptoethanol. Samples were boiled for 5 minutes and separated on a 12% SDS-PAGE electrophoresis gel (20 μl per lane). After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore; Billerica, Mass.) using wet transfer cell system (Bio-rad; Hercules, Calif.). The membrane was blocked for two hours in PBS containing 5% skim milk. TNF-α and caspase-3 expression were detected by incubating the membrane with a primary antibody, rabbit anti-human TNF-α and rabbit anti-human caspase-3 respectively (1:2000 dilution, Abcam; Cambridge, United Kingdom) overnight at 4° C. The primary antibody solutions were discarded and the membrane was incubated for two hours with a solution containing a secondary antibody enzyme horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (1:1000 dilution, Abcam). The TNF-α protein and caspase-3 protein were detected by enhanced chemiluminescence (ELC, Thermo Scientific™ Pierce; Lenexa, Kans.). An anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Santa Cruz Biotechnology; Dallas, Tex.) was used as a protein loading control.
5
Cytochrome c Release in HCC Cells
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HepG2, Hep3B, Huh7 and SNU449 were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum. Chang and Huh1 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum. To detect cytochrome c release, we prepared cytosolic and mitochondrial lysates of the cells. Briefly, the HCC cells were homogenized, and mitochondrial fractions were acquired by a mitochondria isolation kit (Thermo Fisher Scientific, Rockford, IL, USA). The primary antibodies used include: SAG polyclonal antibody (Abcam, Cambridge, MA, USA), Bcl-2 monoclonal antibody (Santa Cruz, Dallas, TX, USA), Bax polyclonal antibody (Cell Signaling, Danvers, MA, USA), Noxa polyclonal antibody (Santa Cruz), SARM monoclonal antibody (Cell Signaling), ubiquitin monoclonal antibody (Santa Cruz), GAPDH monoclonal antibody (Santa Cruz), VDAC polyclonal antibody (Cell Signaling), cytochrome c monoclonal antibody (BD Biosciences, San Jose, CA, USA), Alexa 488-conjugated secondary antibody (Invitrogen, Grand Island, NY, USA) and a loading control, β-actin (Sigma, St Louis, MO, USA).
6
Quantifying Lung Fibroblast Extracellular Matrix Proteins
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Conditioned media and cell lysates from cultured primary lung fibroblasts were analyzed by immunoblotting. The following antibodies were used: fibronectin (FN) monoclonal antibody (clone EP5) (Santa Cruz, Dallas, TX, USA), collagen alpha (I) (Col1α1) polyclonal antibody (Abnova, Taipei City, Taiwan), collagen alpha (III) (Col3α1) polyclonal antibody (Novus Biologicals, Centennial, CO, USA), human IGFBP-5 polyclonal antibody (GroPep Bioreagents, Thebarton, SA, Australia), alpha smooth muscle actin (α-SMA) polyclonal antibody (Abcam, Cambridge, UK), proliferating cell nuclear antigen (PCNA) (Abcam, Clone ab29), and GAPDH monoclonal antibody (Santa Cruz) as primary antibodies and horseradish peroxidase-conjugated antibody as a secondary antibody. Signals were detected using chemiluminescence on FluorChem R System (ProteinSimple, San Jose, CA, USA). Densitometry was analyzed with ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).
7
Extracellular Matrix Protein Analysis
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Western blot analysis of fibroblast extracellular matrix fractions was done as previously described (12 (link)). The following antibodies were used: fibronectin (FN) monoclonal antibody (clone EP5), collagen type I (COL) polyclonal antibody, GAPDH monoclonal antibody (Santa Cruz, Dallas, TX, USA), vitronectin (VN) polyclonal antibody (Biogenesis, Poole, UK), and horseradish peroxidase-labeled secondary antibody (Santa Cruz, Dallas, TX, USA). Signals were detected using chemiluminescence (ProteinSimple, San Jose, CA, USA).
8
Western Blot Analysis of Key Proteins
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According to the literature, β-catenin, Axin2 protein, Cyclin D1 and GRG5 antibody (Abcom, Cambridge, MA, USA) were used in Western blot. GAPDH monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a loading control antibody. Proteins were detected using ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences, Piscataway, NJ, USA). The gray values were analyzed using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
9
Immunoblot for Gestational Diabetes Effects
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An immunoblot assay was evaluated for the effect of
GDM on p15 and p16 protein expression in the pancreatic
islets of the offspring. In short, 35 μg of the total proteins
from each control and OGD groups were run on 10%
polyacrylamide gels and transferred to nitrocellulose
membranes sheets using a transblot system (Bio-Rad,
USA). Western blot analysis was performed using the
p15 (Sigma, USA) and p16 (Proteintech, Japan) primary
antibodies. Monoclonal GAPDH antibody was used as
a loading control (Santa Cruz Biotechnology, Japan).
Immune-blot assay kit (Bio-Rad, USA) was used to
visualize the protein bands. After scanning of the blots,
they were quantified using Quantity One Software
(BioRad, USA).
GDM on p15 and p16 protein expression in the pancreatic
islets of the offspring. In short, 35 μg of the total proteins
from each control and OGD groups were run on 10%
polyacrylamide gels and transferred to nitrocellulose
membranes sheets using a transblot system (Bio-Rad,
USA). Western blot analysis was performed using the
p15 (Sigma, USA) and p16 (Proteintech, Japan) primary
antibodies. Monoclonal GAPDH antibody was used as
a loading control (Santa Cruz Biotechnology, Japan).
Immune-blot assay kit (Bio-Rad, USA) was used to
visualize the protein bands. After scanning of the blots,
they were quantified using Quantity One Software
(BioRad, USA).
10
Western Blot Analysis of α-Synuclein
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