Briefly, 20 μg of cellular proteins were subjected to SDS-PAGE (NuPAGE precast gel, Thermo Fisher Scientific, USA) for electrophoresis and followed by dry transfer onto nitrocellulose membranes (iBlot Transfer Stack, Thermo Fisher Scientific, USA). The membranes were blocked with 5% BSA in TBS containing 0.1% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 60 minutes and incubated with the following primary antibodies: anti-human SIRT1 (1:2000 dilution), acetylated-p53 at K382 (Ac-p53; 1:500 dilution), AMPKα (1:4000 dilutuion), and p21 (1:2000 dilution) antibodies purchased from Cell Signaling Technology (Beverly, MA, USA), and p300 (1:2000 dilution) and total p53 (1:2000 dilution) from Santa Cruz (Dallas, USA). Anti-human GAPDH (1:5000 dilution), and β-actin (1:10000 dilution) antibodies were used as loading controls and purchased from Millipore (Billerica, USA) and Sigma Aldrich (St Louis, USA), respectively. Anti-rabbit and anti-mouse secondary antibodies (1:10000 dilutions) were bought from GE Healthcare (Buckinghamshire, United Kingdom). The immunoreactive bands were detected by an enhanced chemiluminescence system (Proteinsimple, Santa Clara, USA). Related signals were quantified using Proteinsimple image software (Santa Clara, USA).
Anti human gapdh
Manufactured by Merck Group
Sourced in United States
The Anti-human GAPDH is a laboratory reagent used for the detection and quantification of the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein in human samples. GAPDH is a key enzyme involved in the glycolytic pathway and is commonly used as a reference or housekeeping gene in various biological studies. The Anti-human GAPDH provides a reliable tool for researchers to measure GAPDH levels, which can be useful in various applications, such as gene expression analysis, protein quantification, and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Iblot transfer stack, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Total p53, by Santa Cruz Biotechnology (1 mentions)
Anti mouse and anti rabbit secondary antibodies, by GE Healthcare (1 mentions)
Nupage precast gel, by Thermo Fisher Scientific (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Anti pparγ, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Anti akr1c3, by Merck Group (1 mentions)
Gel doc analysis system, by Bio-Rad (1 mentions)
Cellstripper, by Corning (1 mentions)
Growth factor reduced matrigel matrix, by Corning (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
Alexa fluor dyes, by Jackson ImmunoResearch (1 mentions)
Poly d lysine hydrobromide, by Merck Group (1 mentions)
Ve cadherin, by Merck Group (1 mentions)
7 protocols using anti human gapdh
1
Quantification of SIRT1-Dependent p53 Acetylation
2
Cell Line Preparation and Antibody Acquisition
3
Western Blot Protein Expression Analysis
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Cultured cells were washed once with PBS buffer and then lysed in lysis buffer for 5 min. The sample was placed on ice for 30 min, and then centrifuged at 12,000 ×g for 30 min. We used the Bio-Rad DC protein assay to determine the protein concentration of each sample. Equal amounts of protein were separated by sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. We washed the PVDF membranes in Tween and Tris-Buffered Saline (TTBS) (20 mM Tris, 0.5 M NaCl, 0.05% Tween-20) for 10 min, and then blocked the membranes in 10% skim milk for 1 h. The primary antibodies were diluted with TTBS as follows: anti-AKR1C3 (1:4000, A6229–200U; Sigma-Aldrich Co.), anti-PPARγ (1:1000; Sigma-Aldrich Co.), β-actin (1:10,000), and anti-human GAPDH (1:3000). We used the Gel Doc analysis system (Bio-Rad) to scan the gray level of each band. Each Western blot was repeated at least twice.
4
Glioblastoma Cell Culture and Invasion Assay
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The antibodies used include anti-human GAPDH (Sigma-Aldrich, Cat# HPA061280, RRID:AB_2684463 ), VE-Cadherin (Sigma, Cat# MABT134), CD97 (Abcam, Cat# ab108368), mouse FLAG (Cell Signaling Technology, Cat# 8146; RRID:AB_10950495 ), rabbit FLAG (Cell Signaling Technology, Cat# 14793), GAPDH (Thermo Fisher Scientific, Cat# AM4300, RRID:AB_2536381 ), rabbit anti-human GAPDH (Sigma-Aldrich, Cat#ZRB374), Ki67 (Thermo Fisher Scientific, Cat# RM-9106, RRID:AB_2341197 ). All secondary antibodies were conjugated to Alexa Fluor dyes purchased from Jackson Immuno Research. Growth factor–reduced Matrigel matrix, (Corning, Cat# 354230), chondroitin sulfate (#230699), and hyaluronic acid (H7630) were used for invasion assay at specified concentrations. To culture primary GBM cells, vessels were coated with 0.1 mg/ml poly D-lysine hydrobromide (Sigma, Cat# P7886) and 0.01 mg/ml laminin (Corning, Cat# 354232). HUVEC cells (Lonza, Cat# C2519A) were cultured in flasks coated with 0.1% gelatin (Sigma, Cat# G9391). HUVEC authentication was done by the supplier with double immunostaining for CD31/CD105 markers and was more than 90% positive. Cells were lifted with either cell stripper (Corning, Cat# 23-25-056-CI), accutase (Sigma, Cat# A6964), or TrypLE (Gibco, Cat# 1349171).
5
Establishing Stable Cell Lines for lncRNA NKILA
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HepG2 (77400), Huh7, HepAD38, HepG2.2.15, and HEK293T (CRL-11268) cells were purchased from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated foetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 mg/mL) in a 5% CO2 incubator at 37 °C. Stable cell lines with over- or low-expression lncRNA NKILA cultured with puromycin selection were constructed using a lentivector-mediated gene transfer system, and the cells with the vector pLVX or pLKO.1 were served as a blank control. The following antibodies were used: mouse anti-myc MAb (05-724; Millipore, Burlington, MA, USA), goat anti-human histone polyclonal (A0150240; Genscript, Piscataway, NJ, USA), rabbit anti-human IκBα polyclonal (9242; Cell Signalling Technology, MA, USA), mouse anti-phospho-IκBα (Ser32/36; 9246; Cell Signalling Technology, MA, USA), mouse anti-tubulin (ab11323; Abcam, Cambridge, MA, USA), mouse anti-FLAG (F1804; Sigma), rabbit anti-GFP (A-21311, Invitrogen), and anti-human GAPDH (G8795; Sigma) antibodies.
6
Protein Extraction and Western Blot Analysis
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Cells were lysed in protein solubilization buffer (PSB) and analyzed by Western blotting as previously described [54 (link)]. Antibodies used were anti-human STAT5 (Millipore), anti-phospho-STAT5 (Cell Signaling), anti-phospho-tyrosine (4G10, Millipore), anti-ABL (8E9, BD Biosciences), anti-phospho-CRKL (Cell Signaling), and anti-human GAPDH (Sigma Aldrich).
7
Western Blotting of Claudin-4 Protein
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Proteins for western blotting were extracted using lysis buffer (Cell RIPA lysis plus protease inhibitor cocktail, Sigma-Aldrich, Dorset, UK) and sonicated (100W, 10s; Microson, New York, USA), then centrifuged (13000xg, 10min, 4°C). Total protein (20μg) was boiled at 95-100°C for 5 min, cooled on ice before electrophoresis on 12% SDS-Tris-Glycine gels and then transferred to a Polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences, Bucks, UK). After overnight rabbit polyclonal claudin-4 Ab (1:1000) incubation (4°C), the membrane was washed repeatedly in tris-buffer saline (TBS) and then incubated for 1h with horseradish peroxidase (HRP; 1:10; Supersignal West Pico rabbit kit (Pierce Fast; Thermo Scientific, Loughborough). Recombinant human claudin-4 was used as a positive control. To confirm equal protein loading, the membrane was stripped and Re-Blot plus strong solution (Millipore, Livingston, UK) was added for 20min.
After washes in TBS, the membrane was incubated at 4°C with anti-human GAPDH (1:5000 dilution, Sigma-Aldrich, Dorset, UK) for 1hr and re-developed.
After washes in TBS, the membrane was incubated at 4°C with anti-human GAPDH (1:5000 dilution, Sigma-Aldrich, Dorset, UK) for 1hr and re-developed.
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