Total proteins were extracted from tissues and cells using a modified RIPA buffer (150 mM NaCl, 1% NP240, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 50 mM Tris pH 7.4) in the presence of Complete Mini protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Western blot analysis was performed after separation of 50 μg of proteins on a SDS/4%–20% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) with conventional methods and an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ, USA). After that, filters were probed with rabbit antihuman Paxillin antibody (1:500, Cell Signaling Technologies, Danvers, MA, USA) and rabbit antihuman GAPDH (1:1,000; Santa Cruz Biotechnology, USA) plus horseradish peroxidase (HRP)-conjugated corresponding secondary antibodies at 1:1,000 (Cell Signaling Technologies). The densities of bands were measured using a computer-assisted image analysis system (Adobe Systems, San Jose, CA, USA) and were normalized against the expression level of GAPDH protein. All experiments were performed on three separate times.
Sds 4 20 polyacrylamide gel
Manufactured by Bio-Rad
Sourced in United States
The SDS/4%–20% polyacrylamide gel is a laboratory equipment used for protein electrophoresis. It is a pre-cast gel that provides a gradient of 4% to 20% polyacrylamide concentration to facilitate the separation of proteins based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence detection kit, by Cytiva (2 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Rabbit anti human gapdh, by Santa Cruz Biotechnology (1 mentions)
Computer assisted image analysis system, by Adobe (1 mentions)
Rabbit anti gapdh, by Merck Group (1 mentions)
Mini protease inhibitor cocktail, by Roche (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Las4000, by GE Healthcare (1 mentions)
Edta free protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Chemi lumi one super, by Nacalai Tesque (1 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (1 mentions)
Ab290, by Abcam (1 mentions)
4 protocols using sds 4 20 polyacrylamide gel
1
Protein Extraction and Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from human HCC cells by using a modified radioimmunoprecipitation assay buffer (150 mM NaCl, 1% NP240, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 7.4) in the presence of cOmplete, Mini protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Western blot analysis was performed after the separation of 50 μg of proteins on a sodium dodecyl sulfate (SDS)/4%–20% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) with conventional methods and an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ, USA). After that, filters were probed with mouse anti-hPTPRJ 1:100, hamster anti-mouse PTPRJ 1:1,000, and rabbit anti-GAPDH (Sigma-Aldrich Co., St Louis, MO, USA) 1:1,000 plus HRP-conjugated corresponding secondary antibodies at 1:1,000 (Cell Signalling Technologies, Danvers, MA, USA). Signals were visualized with SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific) by exposure to films.
3
Testis Protein Extraction and Western Blot
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Testes lysates were prepared in a solution of 150 mM NaCl, 1% Nonidet P-40, 10 mM Tris–HCl (pH 8.0) containing EDTA–free protease inhibitor cocktail (Nacalai) and phenylmethylsulfonyl fluoride. The soluble materials were resolved on SDS–4–20% polyacrylamide gel (Bio-Rad Lab, Berkeley, CA, USA) and transferred onto a polyvinylidene difluoride membrane (Bio-Rad Lab). The filters were blocked with 5% skim milk in PBS containing 0.1% Tween20 and then sequentially incubated with primary and secondary antibodies. The blots were developed by Chemi–Lumi One Super (Nacalai) and signals were detected by LAS4000 (GE Healthcare, Chicago, IL, USA). For the detection of USP26, USP26 was immunoprecipitated from testes lysates with anti–USP26 antibody Ab2 (see Antibodies) and the precipitated proteins were subjected to Western blot analysis.
4
Chromatin-Associated Protein Identification
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Transduced 3T3 fibroblasts containing mEGFP, mEGFP-Cbx2, or mEGFP-Cbx2-23KRA were grown to 80% confluency in 15-cm tissue culture dishes. Medium containing 500 ng/mL doxycycline was added for 24 h. Cells were washed with PBS and collected using a cell scraper. Nuclear extracts were prepared as described previously (Abmayr et al. 2006 (link)). Protein levels in nuclear extracts were measured on a Nanodrop using A280. Equal protein mass between samples was used in subsequent co-IPs. One percent volume was saved as input. For co-IP, magnetic protein A beads (Invitrogen) were pre-equilibrated in BC buffer containing 300 mM KCl and 0.05% NP-40. Washes were performed on a magnetic rack. For each immunoprecipitation, 2.5 µg of GFP antisera (Abcam, ab290) was conjugated to pre-equilibrated beads by incubating for 1 h at 4°C. GFP antisera-conjugated beads were washed three times with BC buffer containing 300 mM KCl and 0.05% NP-40 and mixed with nuclear extracts for 2 h at 4°C. Immunoprecipitations were washed three times with BC buffer containing 300 mM KCl and 0.05% NP-40 and resuspended in 1× SDS sample buffer. Samples were heated for 5 min to 95°C, and supernatant was loaded onto an SDS 4%–20% polyacrylamide gel (Bio-Rad). Samples were processed for either mass spectrometry or immunoblotting.
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