The proteins were extracted from cells or tissues using RIPA buffer (Beyotime) with protease inhibitors. The BCA Kit was applied to the detection of protein concentrations. Then equal amounts (20 μg) of proteins denatured by boiled water bath were separated by SDS-PAGE and then transferred onto 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in TBST with 5% non-fat milk for 1 h and then incubated with rabbit anti-human primary antibodies against HK2 (ab227198, 1:5000 dilution, Abcam, Cambridge, MA, USA), RPN2(ab244399, 1:2000 dilution, Abcam) or β-actin(ab227387, 1:10000 dilution, Abcam) as a loading control at 4 °C overnight and goat anti-rabbit secondary antibody (ab97051, 1:10000 dilution, Abcam) at room temperature for 2 h. The signals were developed using ECL Kit (Beyotime) and the relative protein levels of HK2 and RPN2 were normalized to the control group.
Ab244399
Manufactured by Abcam
Sourced in United States
Ab244399 is a primary antibody produced in rabbit. It is designed for use in immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab227198, by Abcam (1 mentions)
Ab227387, by Abcam (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Ab97051, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl western blotting substrate, by Promega (1 mentions)
Ab134175, by Abcam (1 mentions)
Exkine total protein extraction kit, by Abbkine (1 mentions)
Ab76151, by Abcam (1 mentions)
Ab39688, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
3 protocols using ab244399
1
Western Blot Analysis of Protein Levels
2
Glioma Cell Line Protein Extraction and Detection
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Briefly, total protein of glioma cell lines was extracted using the ExKine Total Protein Extraction Kit (Abbkine) in accordance with its protocol. Equal amounts of protein (30 μg/lane) were separated by 10% SDS‐PAGE and subsequently transferred to PVDF membranes (EMD Millipore). After blocking for nonspecific binding, the membranes were incubated with antibodies for RPN2 (1:200 dilution; ab244399; Abcam), TCF4 (1:10,000 dilution; ab76151; Abcam), c‐myc (1:1000; ab39688; Abcam), cyclin D1 (1:10,000; ab134175; Abcam) or β‐actin (1:5000; ab6276; Abcam) overnight at 4°C and followed by incubation with secondary antibodies for 1 h at room temperature. Eventually, an ECL western blotting substrate (Promega) was used to develop the protein bands.
3
Immunohistochemical Analysis of RPN2 and β-Catenin
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Sections underwent dewaxing, re‐hydration, antigen retrieval, and blocking, and then were incubated with antibodies against RPN2 (1:200; ab244399; Abcam) and β‐catenin (1:500; ab32572; Abcam) overnight at 4°C and then washed three times with PBST. Sections were incubated with HRP‐conjugated secondary antibody for 15 min at room temperature and then stained with diaminobenzidene (DAB) and hematoxylin.
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