Cell lysates were rinsed with ice-cold PBS and prepared using lysis buffer with 1% Triton, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), and 1% deoxycholate and protease inhibitors cocktail (Roche). Protein concentration was determined by Bradford protein assay kit (Bio-Rad) and equal amount of protein was separated by 12% SDS-PAGE using Bio-Rad apparatus. The membranes were blocked for 2 hours in 5% skim milk at room temperate after protein was transferred to PVDF membrane (Millipore, Billerica, MA, USA). The proteins were incubated with the following primary antibodies: IL-6 and TNF-α (Merck Millipore, Bedford, MA, USA), ICAM-1, Hes5 and MCP-1 (Abcam, Cambridge, Mass, USA), Hes1 (OriGene Technologies, Rockville, MD, USA), Hey1 and Hey2 (Proteintech Group, Chicago, IL, USA), and phospho-p65 (CST, Chicago, IL, USA). Anti-GAPDH antibody (Bioworld, Nanjing, China) was used as the loading control in all western blots. The secondary antibodies, anti-mouse and anti-rabbit IgGs conjugated to horseradish peroxidase, were obtained from Kangchen (Shanghai, China). Finally, protein bands were visualized using Supersignal West Femto Substrate (Pierce, Rockford, IL, USA).
Anti gapdh antibody
Manufactured by Bioworld Technology
Sourced in United States, United Kingdom, China
The Anti-GAPDH antibody is a laboratory tool used to detect the presence and quantify the levels of the GAPDH protein in biological samples. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a widely expressed and highly conserved enzyme involved in the glycolytic pathway. This antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression and distribution of GAPDH in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Pvdf membrane, by Abcam (2 mentions)
Il 6 and tnf α, by Merck Group (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Ecl detecting kit, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Proteintech (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Antibodies against phospho akt ser473, by Cell Signaling Technology (1 mentions)
Biospectrum 600 imaging system, by Analytik Jena (1 mentions)
Phospho akt thr308, by Santa Cruz Biotechnology (1 mentions)
Mmp2 and mmp9 antibodies, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Anti gata 1 antibody, by Abcam (1 mentions)
23 protocols using anti gapdh antibody
1
Western Blot Analysis of Inflammatory Mediators
2
Western Blot Analysis of Thyroid Proteins
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Total proteins were extracted from cells or tissues using a radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing proteinase inhibitors. A bicinchoninic acid (BCA) assay (Thermo, Rockford, IL, USA) was performed to measure protein concentrations. 30 μg of extracted proteins per well was loaded onto 8–12% SDS-PAGE gel (Beyotime, Shanghai, China) for electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The PVDF membranes were blocked with 5% milk solution for 2 h and then incubated with primary antibody diluted in 5% bovine serum albumin (Sigma, St Louis, MO, USA) overnight at 4°C. After that, membranes were washed in 0.1% PBS/Tween-20 (PBST) and probed with secondary antibody for 1.5 h at room temperature. After being washed 3 times in PBST again, the membranes were visualized using the ChemiDoc XRS System (BioRad, Hercules, CA) by enhanced chemiluminescence (ECL) detecting kit (Thermo, Rockford, IL, USA). Antibodies applied in this study were anti-TNRC6C antibody (Santa Cruz, sc-244474), anti-TSHR antibody (Proteintech, 14450-1-AP), anti-TPO antibody (Affinity, DF8279), anti-Pendrin antibody (Santa Cruz, sc-50346), anti-NIS antibody (Affinity, DF2242), anti-GAPDH antibody (Bioworld, AP0063) and anti-β-actin antibody (Proteintech, 60008-1-IG).
3
Western Blotting Analysis of Protein Expressions
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Western blotting was conducted after separation of polypeptides using SDS-PAGE. Proteins on gel were transferred to PVDF membrane (Merck Millipore; Billerica, MA, USA). The membrane was further incubated with indicated primary and appropriate secondary antibodies. Antibodies against ACOX1 were obtained from Proteintech Group (Chicago, IL, USA). Phosphorylated form of ERK1/2 and ERK1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BD Biosciences, respectively. Antibodies targeting MMP3, MMP10 and phospho-Akt (Thr308) were also purchased from Santa Cruz Biotechnology. Antibodies against phospho-Akt (Ser473) were obtained from Cell Signaling Technology (Danvers, MA, USA). MMP2 and MMP9 antibodies were purchased from Abcam (Cambridge, Cambridgeshire, UK). An anti-GAPDH antibody (BioWorld; St. Louis Park, MN, USA) was used as control. HRP-conjugated secondary antibodies against rabbit IgG or mouse IgG (GeneTex) were incubated with membrane for 1 h at room temperature. Immunobands were detected using chemiluminescent HRP substrates (ECL; Merck Millipore) and captured by UVP BioSpectrum 600 Imaging System. The intensity of bands was quantified by Image J software.
4
Splenic B Cell LPS Stimulation
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Splenic B lymphocytes were isolated and treated with LPS (concentration 1 µg/ml, Sigma, St Louis, MO, USA) for 24 h. Proteins were electrophoresed by 10% SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride membrane. After blocking for 2 h with phosphate-buffered saline containing 0.1% Tween 20 and 5% powdered skim milk, the membrane was incubated with anti-NF-κB p-65 antibody and anti-TLR4 antibody (1:1000 Abcam, Cambridge, UK), anti-GAPDH antibody (1:2000 Bioworld, Minnesota, USA) overnight in 5% powdered skim milk buffer. Then the blots were washed and incubated with secondary antibodies. All bands were detected using the enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK) and analyzed by LabWorks (TM ver4.6,UVP, BioImaging systems).
5
Protein Expression Analysis of TSA and VPA
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Cells were treated with TSA or VPA at different concentration for 48 h. Total protein was extracted from cells and LADC tissue with lysis buffer containing protease inhibitor cocktail (Keygentec, China) and the concernrations were determined by the BCA Protein Assay Kit (Beyotime). Equal amount of protein were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore). The membrane were blocked with 5% nonfat milk in TBST (0.1% Tween-20) and then incubated with anti-IRF-3 antibody (Abcam), anti-GATA-1 antibody (Abcam), or anti-GAPDH antibody (Bioworld) at 4 °C overnight at a dilution of 1:2000-1:4000, followed by anti-rabbit IgG (Cell signaling) or anti-mouse IgG (Jackson ImmunoResearch Laboratories) at a dilution of 1:5000. Intensity of bands was measured using an ECL imager.
6
Western Blot Analysis of EMT Markers
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Whole-cell lysates were collected using RIPA buffer. Proteins were separated using 10% SDS polyacrylamide gel, and the gels were transported to PVDF membranes (Thermo Fisher Scientific, CA, USA). The PVDF membranes were incubated with 5% skim milk in TBST at room temperature for 1 h. Later on, the PVDF membranes were probed with primary antibodies: anti-DCLK1 antibody (1:1000, Abcam, CA, USA), anti-Notch1 antibody (1:3000, Abcam, CA, USA), anti-E-cadherin (1:3000, Abcam, CA, USA), anti-Vimentin (1:3000, Abcam, CA, USA), anti-slug (1:1000, Abcam, CA, USA), anti-TGF-β (1:1000, Abcam, CA, USA), anti-MMP2 (1:1000, Abcam, CA, USA), anti-MMP9 (1:1000, Abcam, CA, USA) and anti-GAPDH antibody (1:3000, Bioworld, CA, USA) overnight at 4 °C. After that, the PVDF membrane was incubated for 1 h in secondary antibody anti-rabbit IgG second antibody (Abcam; ab150077) (1:5000) at room temperature for 1 h. Finally, the immunoreactivity was detected using ECL reagent (Santa Cruz Biotechnology).
7
Western blot analysis of protein expression
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Total protein was extracted, separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat dry milk in TBST and were incubated with following primary antibodies: anti-MNAT1 antibody, anti-EIF2A antibody, anti-KSR1 antibody, anti-ADCY9 antibody and anti- kLRP1 antibody (1:1000 Abcam, Cambridge, UK), anti-PAM16 antibody, anti-SF3A3 antibody, anti-LSM5 antibody and anti-TRAM2 antibody (1:800 Abcam, Cambridge, UK), anti-BMP6 antibody (1:400 Abcam, Cambridge, UK), anti-GAPDH antibody (1:2000 Bioworld, Minnesota, USA) at 4 °C overnight. Secondary antibodies were added, and the bands were visualized using the enhanced chemiluminesczence (Amersham Biosciences, Buckinghamshire, UK). GAPDH was used as the loading control.
8
Protein Expression Analysis of Cellular Signaling
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Total protein from the treated cells and tissues were extracted by RIPA buffer with the mixture consisting of protease Inhibitor Cocktail (Beyotime, Shanghai, China). Protein was separated by an SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, IPVH10100). After blocking with 5% bovine serum albumin (BSA) for 75 minutes, the PVDF membrane was incubated with primary antibodies (YAP Rabbit mAb (1:1000, CST, #14074), TEAD1 Rabbit mAb (1:1000, CST, #12292), Anti-Ki67 Rabbit pAb (1:1000, Servicebio, GB11030), NF-κB p65 (D14E12) XP® Rabbit mAb (1:1000, CST, #8242), Anti-Cyclin D1 antibody (1:1000, Abcam, ab16663), Anti-CDK4 antibody (1:1000, Abcam, ab108357, Phospho-IκBα (Ser32/36) (5A5) Mouse mAb (1:1000, CST, #9246), IκBα (112B2) Mouse mAb (Carboxy-terminal Antigen, 1:1000, CST, #9247) and rabbit monoclonal anti-GAPDH antibody (1:3000, Bioworld, AP0063) at 4 °C overnight. After washing for three times, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, enhanced chemiluminescence (Advansta, K-12045-D50) was used to visualize the immunoblot. The intensity of the bands was determined by Image J software. The ratio of each band/GAPDH was considered as the expression level of the target protein.
9
Western Blot Analysis of Apoptosis Markers
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After exposure to different treatments, cells were collected and lysed in ice-cold buffer (tris-(hydroxymethyl) aminomethane 50 mmol/l, pH 7.4, NaCl 150 mmol/l, 0.5% Triton X-100, edetic acid 1 mmol/l, phenylmethylsulfony fluoride 1 mol/l, and aprotinin 5 mg/l), and then centrifuged at 12,000 g for 5 min at 4°C. The supernatant was collected and the protein concentration was determined using the BCA-100 Protein Quantitative Analysis Kit (Beyotime, Haimen, China). Equal amounts of proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA) followed by blocking in 5% non-fat milk for 1 h. The membranes were then incubated overnight at 4°C with rabbit polyclonal anti-Bcl-2, rabbit polyclonal anti-Bax, rabbit monoclonal anti-PARP, rabbit monoclonal anti-caspase-3 or rabbit polyclonal anti-iNOS antibodies (1∶1000, Cell Signaling Technology, Beverly, MA). After washing with TBS/T (TBS with 0.05% Tween 20), membranes were incubated in IRDye 680-conjugated affinity purified goat anti-rabbit IgG (1∶5000, Bioworld Technology, Inc., Minneapolis, MN, USA)) at room temperature for 1 h. The images were scanned with the Odyssey infrared imaging system (LI-COR) and quantified with Multi Gauge. GAPDH (Anti-GAPDH antibody, Bioworld, Minneapolis, MN, USA), was used as an internal reference.
10
Quantifying Aortic Protein Expression
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The aortas were frozen in liquid nitrogen and homogenized in a cell extract denaturing buffer that contained a phosphatase inhibitor cocktail and protease inhibitor cocktail. The cultured cells were resolved using RIPA Lysis Buffer. The antibodies for ET A R (Abcam, ab85163), ET B R (Abcam, ab117529), phospho-NF-κB p65 (Abcam, ab86299), and NF-κB p65 (Abcam, ab16502) were used as primary antibodies. Secondary antibodies were HRP-labeled goat anti-rabbit IgG or rabbit anti-goat IgG (Biosharp). All antibodies were used at a dilution of 1:1000, and the experiments were repeated 3 times. An anti-GAPDH antibody (Bioworld, AP0063) was used for normalization. The western blot band intensity was determined using IMAGE-PRO PLUS.
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