Western blots were carried out using the following antibodies at the indicated dilutions: αTcf7l1 (Santa Cruz sc8635, 1:500), αcMyc (Santa Cruz sc42, 1:500), αp‐cMycSer62 (Abcam ab185656, 1:1,000), αp‐cMycThr58 (Santa Cruz sc135647, 1:500), alpha‐tubulin (Abcam ab126165, 1:1,000), αgoat HRP (Abcam ab6741, 1:2,000), αmouse HRP (Abcam ab6728, 1:2,000), and αrabbit HRP (Abcam ab16284, 1:2,000).
Ab126165
Manufactured by Abcam
Sourced in United States
Ab126165 is a laboratory instrument designed for the detection and quantification of specific biomolecules. It utilizes an advanced analytical technique to provide reliable and accurate measurements. The product's core function is to facilitate the analysis of samples, enabling researchers to gather valuable data for their scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Ab6741, by Abcam (1 mentions)
Ab16284, by Abcam (1 mentions)
Ab6728, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Anti mmp2, by Cell Signaling Technology (1 mentions)
Anti mmp9, by Cell Signaling Technology (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab86607, by Abcam (1 mentions)
Ab28340, by Abcam (1 mentions)
Af3715, by R&D Systems (1 mentions)
Ab65869, by Abcam (1 mentions)
5 protocols using ab126165
1
Antibody-Based Western Blot Analysis
2
Western Blot Analysis of ISG15
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Cells were harvested and lysed in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 3% sodium dodecyl sulfate (SDS), 10% glycerol, 50 mM DL-dithiothreitol (DTT), and 0.1% bromophenol blue) with protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentrations were determined by the BCA method (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA). The proteins (10 ug) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Bovine serum albumin (5%) in TBS-T (1 mol/L Tris-HCl (pH 7.5), 0.8% NaCl and 0.1%Tween 20) was used to block the membrane. Then, the membrane was incubated with anti-ISG15 (Abcam, ab131119, Cambridge, MA, USA), anti-α-tubulin (Abcam, ab126165, Cambridge, MA, USA), and anti-β-actin (Sigma-Aldrich, A5441, St.Louis, USA) antibodies at 4°C overnight. The blots were then treated with an HRP-conjugated secondary antibody (Pierce, Rockford, IL, USA).
3
Protein Extraction and Western Blot Analysis
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Tissue protein lysis was prepared as described (Wang et al, 2009 (link)). Cell lysates were generated with RIPA lysis buffer (Guangzhou Fans Biotechnology, Co. Ltd., Guangzhou, China). Nuclear protein was extracted by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA). Protein concentration was determined by a standard BCA assay, and 60 μg total cell lysates or nuclear extracts were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred onto PVDF membranes (Amersham Biosciences, Buckinghamshire, UK), and membranes were probed with anti-Snail1 (sc-28199; Santa Cruz Biotechnology), anti-MMP2 (#13132), anti-MMP9 (#3852; Cell Signaling), anti-COUP-TFII (PP-H7147-00; Perseus Proteomics, Tokyo, Japan), all in 1 : 1000 dilution, or anti-tubulin (ab126165; Abcam, Cambridge, MA, USA) (1 : 2000 dilution) and visualised with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and ECL kits (Amersham Pharmacia Biotech, Buckinghamshire, UK). Protein bands were quantified by Total lab software (Nonlinear Dynamics, Newcastle upon Tyne, UK).
4
Western Blot Analysis of Cell Proteins
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Total proteins of cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After being blocked with 5% non-fat dry milk in PBS, the membrane was incubated with antibodies to FAPα (1:1,000, AF3715; R&D Systems, Inc., Minneapolis, MN, USA), dipeptidyl peptidase 4 (DPP4; 1:1,000, ab28340; Abcam, Cambridge, MA, USA) or matrix metalloproteinase (MMP)2 (1:1,000, ab86607; Abcam) at 4°C overnight. After being washed several times, the polyvinylidene difluoride membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 hours. The bands were then detected by Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer’s protocols. β-Tubulin protein levels were also determined by using the specific antibody (1:3,000, ab126165; Abcam) as a loading control.
5
Western Blot Analysis of MIF Protein
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Nasopharyngeal carcinoma cells were harvested and lysed in sodium dodecylsulfate (SDS) sample buffer. Cellular proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Then, the membranes were incubated with MIF (1:1000, Abcam, ab65869) and α-tubulin (1:3000, Abcam, ab126165) antibodies at 4 °C overnight. At last, the blots were treated with a horseradish peroxidase (HRP)-conjugated secondary antibody.
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