Gapdh ab181602

Manufactured by Abcam

Sourced in United States, United Kingdom

GAPDH (ab181602) is a recombinant protein that functions as the glyceraldehyde 3-phosphate dehydrogenase enzyme. This enzyme is involved in the glycolytic pathway and catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate.

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Lab products found in correlation

Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bio spectrum gel imaging system, by Bio-Rad (1 mentions) Tris buffered saline tween tbst, by Boster Bio (1 mentions) β actin ab179467, by Abcam (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Ab32389, by Abcam (1 mentions) P mtor ab109268, by Abcam (1 mentions) Quantity one, by Thermo Fisher Scientific (1 mentions) Nupage running buffer, by Thermo Fisher Scientific (1 mentions) Hrp labeled goat anti mouse igg h l a0216, by Beyotime (1 mentions) Hrp labeled goat anti rabbit igg h l a0208, by Beyotime (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Chemidoc touch, by Bio-Rad (1 mentions) Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions) Ab8932, by Abcam (1 mentions) Ab8805, by Abcam (1 mentions) Ab2732, by Abcam (1 mentions) Anti rabbit or anti mouse igg horseradish peroxidase hrp linked secondary antibodies, by Cell Signaling Technology (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)

13 protocols using gapdh ab181602

Western Blot Analysis of Exosomal Markers

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The extraction of the total protein was performed via radio-immunoprecipitation assay lysis buffer embodying phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, Shanghai, China). The protein level in the supernatant was detected via the BCA method. Equal volumes of protein (50 mg) were separated via SDS-PAGE (10% gel) and then transferred onto the polyvinylidene fluoride (PVDF) membrane (EMD Millipore). The PVDF membranes were incubated with tris-buffered saline tween (TBST; Boster Biological Technology Co., Ltd.) supplemented with 5% skimmed milk to block the non-specific binding. Next, the membranes were cultured with the primary antibodies (Table 1) at 4°C overnight, together with rabbit anti-rat secondary antibody for 1 h at room temperature. The proteins were developed in enhanced chemiluminescence reagent, and analyzed by BioSpectrum gel imaging system (Bio-Rad Laboratories, Inc.).Table 1

Antibodies for Western Blot Analysis

Antibody	No., Company	Dilution Ratio
Alix	ab117600, Abcam	1: 100
CD63	ab217345, Abcam	1: 100
CD9	ab92726, Abcam	1: 100
CD81	ab79559, Abcam	1: 5000
GAPDH	ab181602, Abcam	1: 50
β-actin	ab179467, Abcam	1: 5000
Cleaved caspase-3	ab2302, Abcam	1:50
Cleaved PARP	ab32064, Abcam	1: 5000
Secondary antibody	ab150117, Abcam	1: 5000

Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PARP, poly (ADP-ribose) polymerase; Abcam, Abcam Inc., Cambridge, MA, USA.

Bu X., Li D., Wang F., Sun Q, & Zhang Z. (2020). Protective Role of Astrocyte-Derived Exosomal microRNA-361 in Cerebral Ischemic-Reperfusion Injury by Regulating the AMPK/mTOR Signaling Pathway and Targeting CTSB. Neuropsychiatric Disease and Treatment, 16, 1863-1877.

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Protein Expression Profiling by Western Blot

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Protein lysates were collected from cells that treated by si‐ESCO2 or si‐con, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF; protease inhibitor) was added into RIPA buffer to extract protein successfully. Then, protein was separated in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Finally, the target proteins were probed using primary antibodies and secondary antibodies. All the antibodies including ESCO2 (ab86003), GAPDH (ab181602), AKT (ab179463), p‐AKT (ab38449), mTOR (ab134903), p‐mTOR (ab109268), PCNA (ab92552), and p53 (ab32389) were purchased from Abcam. The protein bands were developed by enhanced chemiluminescence reagent (Thermo Fisher Scientific), and QUANTITY ONE was applied to detect the gray values of proteins.

Wang Q, & Liu L. (2020). Establishment of cohesion 1 homolog 2 facilitates cell aggressive behaviors and induces poor prognosis in renal cell carcinoma. Journal of Clinical Laboratory Analysis, 34(5), e23163.

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Protein Extraction and Western Blot Analysis

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After treatment, cells were lysed by Radio Immunoprecipitation Assay (RIPA) lysis buffer, incubated on ice for 30 min, followed by centrifugation at 12,000 × g for 10 min at 4 ℃. The supernatant was collected as the whole protein extract. The protein extract was denatured by adding NuPAGE running buffer (Thermo Fisher Scientific, Cat# NP0001) and heating up at 100 ℃ for 10 min. Protein samples were stored at − 80 ℃ or used directly for western blotting. Total proteins were electrophoresed on an SDS-PAGE gel for 1.5 h to separate the proteins, which were then transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Cat# 1,620,177). They were then co-incubated with primary antibodies and incubated with secondary antibodies conjugated to horseradish peroxidase (HRP). Antibodies were diluted with 5% bovine serum albumin (BSA). Primary antibodies against DNMT1 (24206-1-AP) and DNMT3A (20954-1-AP), were purchased from proteintech Group (Wuhan, China). Primary antibodies CTCF (ab128873), PAX5 (ab109443) and GAPDH (ab181602) were purchased from Abcam (Cambridge, UK). HRP-labeled Goat Anti-Mouse IgG (H + L) (A0216) and HRP-labeled Goat Anti-Rabbit IgG (H + L) (A0208) were purchased from Beyotime (Guangzhou, China). The HRP signal was detected using ChemiDoc Touch (Bio-Rad).

Wang P., Meng Z., Deng K., Gao Z, & Cai J. (2024). Vpr driving DNA methylation variation of CD4 + T cells in HIV-1 infection. Virology Journal, 21, 97.

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Protein Extraction and Western Blot Analysis of BMSCs

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Extraction of total protein from BMSCs was carried out using Radio-Immunoprecipitation assay lysis buffer covering protease inhibitors (Sigma-Aldrich; Merck KGaA), and protein concentration was measured using Pierce BCA Protein assay Kit (Thermo Fisher Scientific, Inc.). Subsequently, the total protein extract was separated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated with primary antibodies RUNX2 (AB23981) and GAPDH (AB181602) (both 1: 1000, Abcam) and with horseradish peroxidase-conjugated secondary antibody. Visualization of the bands was implemented using an enhanced chemiluminescence reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and analysis was performed using ImageJ software (National Institutes of Health).

Shen Y., Jiang B., Luo B., Jiang X., Zhang Y, & Wang Q. (2023). Circular RNA-FK501 binding protein 51 boosts bone marrow mesenchymal stem cell proliferation and osteogenic differentiation via modulating microRNA-205-5p/Runt-associated transcription factor 2 axis. Journal of Orthopaedic Surgery and Research, 18, 782.

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Evaluation of Anti-Cancer Compounds

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Apatinib mesylate standard products were provided by Jiangsu Heng Rui Pharmaceutical Co. Ltd. (Jiangsu, China) and icotinib standard product was obtained from Zhejiang Beida Pharmaceutical Co., Ltd. (Zhejiang, China), pemetrexed was provided by Qilu Pharmaceutical Co., Ltd. (Shandong, China) and osimertinib standard was purchased from Selleck.cn. Three cell lines A549, HCC827 and H1975 were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI media (HyClone) containing 10% fetal bovine serum (FBS, Gibco),100 U/mL penicillin (HyClone) and 100 ug/mL streptomycin (HyClone). Primary antibodies against AKT(ab8805), phosphor‐AKT(ab8932), ERK(ab54230), phospho‐ERK(ab201015), CD31(ab28364), mTOR(ab2732), phosphor‐mTOR(ab109268) and GAPDH(ab181602) were purchased from abcam (Cambridge, MA, USA). Primary antibodies against ki67 (9449) and anti‐rabbit or anti‐mouse IgG horseradish peroxidase (HRP)‐linked secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Liu M., Wang X., Li H., Xu L., Jing L., Jiang P., Liu B, & Li Y. (2019). The effect of apatinib combined with chemotherapy or targeted therapy on non‐small cell lung cancer in vitro and vivo. Thoracic Cancer, 10(10), 1868-1878.

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Western Blot Analysis of TLR4 and NF-κB

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The protein concentration was determined using the BCA protein concentration detection kit (Aspen, Wuhan, China). Equal amounts of proteins were then separated on 10% SDS-PAGE and electroblotted onto a PVDF membrane. Next, the membrane was incubated overnight at 4°C with the primary antibodies: anti-TLR4 (19811-1-AP, 1:1000; Proteintech, Rosemont, IL, USA), anti-p- NF-κB p65 (#3033, 1:500; Cell Signaling Technology, Danvers, MA, USA), anti-NF-κB p65 (#8242, 1:3000; Cell Signaling Technology), and GAPDH (ab181602, 1:10000; Abcam, Cambridge, UK). After that, the membrane was probed with a secondary antibody (AS1107, 1:10000; Aspen) at room temperature. Subsequently, the bands were developed with an enhanced chemiluminescence detection kit (Aspen ) and the band intensity was analyzed using the AlphaEaseFC software.

He G., He Y., Ni H., Wang K., Zhu Y, & Bao Y. (2023). Dexmedetomidine attenuates neuroinflammation and microglia activation in LPS-stimulated BV2 microglia cells through targeting circ-Shank3/mir-140-3p/TLR4 axis. European Journal of Histochemistry : EJH, 67(3), 3766.

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Analyzing JAK/STAT and TGF-β Pathways in Hut78 Cells

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The human TCL cell line Hut78 was obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal mono-nuclear cells were separated from the peripheral blood of healthy volunteers. The anti-mPGES-1 antibody (#10004350) and mPGES-1 inhibitor CAY10526 (#10010088) were ordered from Cayman Chemical Company (Ann Arbor, MI, USA). Antibodies to JAK2 (#3230), STAT3 (#9139), p-STAT3 (#52075), Akt (#4691), p-Akt (#4060), TGF-β (#3709), Smad (#9523) and p-Smad (#9520) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies to JAK1 (ab133666), GAPDH (ab181602) and Caspase-3 (ab32351) were purchased from Abcam (Cambridge, MA, USA).

Li Y., Qiu M., Li B., Xiao J., Yang W., Xie S., Du Y., Huang K, & Nie D. (2022). Growth of T-cell lymphoma cells is inhibited by mPGES-1/PGE2 suppression via JAK/STAT, TGF-β/Smad3 and PI3K/AKT signal pathways. Translational Cancer Research, 11(7), 2175-2184.

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Cigarette Smoke Extract Exposure in iHBECs

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Immortalised human bronchial epithelial cells (iHBECs; courtesy Dr Shay, University of Texas) were used for cigarette smoke extract (CSE) experiments. iHBECs were cultured in keratinocyte-serum free medium (K-SFM; Gibco, UK) supplemented with 25μg/ml bovine pituitary extract (Gibco, UK), 0.2ng/ml epidermal growth factor (Gibco, UK), 25μg/ml G418 sulphate (VWR Life Science, UK) and 250ng/ml puromycin (MilliporeSigma, UK). ELK1 #ab32106, β-tubulin #ab6046, GAPDH #ab181602, αSMA #ab5694 and NIMP #ab2557 antibodies for western blotting and/or immunohistochemistry (IHC) were from Abcam, UK. CD3+ #MCA1477 and CD68 #OABB00472 antibodies for IHC were obtained from Bio-rad, UK and Aviva Systems Biology, UK, respectively. Reagents required for the synthesis of cDNA from RNA, were supplied from Invitrogen, UK (SuperScript^™ IV Reverse Transcriptase), Qiagen, UK (Nuclease free water), Roche, UK (OligoDT) or Promega, UK (RNasin inhibitor, dNTPs). Tobacco laboratory research grade cigarettes (batch 1R6F) were from the University of Kentucky, USA.

Cairns J.T., Habgood A., Edwards-Pritchard R.C., Joseph C., John A.E., Wilkinson C., Stewart I.D., Leslie J., Blaxall B.C., Sustak K., Alberti S., Nordheim A., Oakley F., Jenkins G, & Tatler A.L. (2019). Loss of ELK1 has differential effects on age-dependent organ fibrosis. The international journal of biochemistry & cell biology, 120, 105668.

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Comprehensive Immunoprecipitation and Western Blot Protocol

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HepG2 cells and SK-HEP-1 cells were lysed using RIPA lysis solution containing complete protease inhibitor and phenylmethylsulfonyl fluoride. The proteins were quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, China). For immunoprecipitation, the cells were collected in RIPA buffer. The resulting lysates were precipitated with the relevant antibody and protein G-sepharose beads by incubation at 4°C overnight. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated with the relevant primary antibody. Immunostaining was detected with a Bio-Rad ChemiDoc XRS imaging system (Bio-Rad, USA).
The following antibodies were used. FAF1 (ab183045), JNK (ab179461), and GAPDH (ab181602) were obtained from Abcam. IRS1 (2382S), AKT (9272S), p-AKT (ser473, 4060S), GSK-3β (12456S), p-GSK-3β (5558S), and p-JNK (4668S) were purchased from Cell Signaling Technology. Flag (66008-3-Ig) and IgG (SA00001-1) were purchased from Proteintech.

Sun B., Zhou J., Gao Y., He F., Xu H., Chen X., Zhang W, & Chen L. (2021). Fas-Associated Factor 1 Promotes Hepatic Insulin Resistance via JNK Signaling Pathway. Oxidative Medicine and Cellular Longevity, 2021, 3756925.

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Comparative Protein Expression Analysis in Gastric Cancer

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The methods were described as previously described [3 (link)]. Anti-GSK-3β (#9832), AKT (pan) (#4685), phosphorylated AKT (Ser473) (#4060), GSK-3β (Ser9) (#12456) and phosphorylated GSK-3β (Ser9) (#5558) were purchased from Cell Signaling Technology. Anti-CDK5RAP3 (ab157203), E-cadherin (ab1416), N-cadherin (ab76057), Vimentin (ab8978), and GAPDH (ab181602) antibodies were purchased from Abcam. AKT siRNA (#6211) and control siRNA (#6568) were purchased from Cell Signaling Technology. We corrected the loading error based on loading controls and compared the expression levels of target proteins in gastric tumor and adjacent non-tumor tissues. The protein expression in tumors was defined as high level when it was higher than that in normal tissue but was defined as low level when it was lower than that in normal tissue.

Zheng C.H., Wang J.B., Lin M.Q., Zhang P.Y., Liu L.C., Lin J.X., Lu J., Chen Q.Y., Cao L.L., Lin M., Tu R.H., Xie J.W., Li P, & Huang C.M. (2018). CDK5RAP3 suppresses Wnt/β-catenin signaling by inhibiting AKT phosphorylation in gastric cancer. Journal of Experimental & Clinical Cancer Research : CR, 37, 59.

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