HEK293 cells were transfected with the indicated constructs for expression of GFP- and Flag-tagged WT and mutant proteins. 24 hours after transfection cells were lysed with 600 μl CHAPS lysis buffer [10 mM 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, Roth), 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM NaF, 1 mM Dithiothreitol (DTT, Merck), 0.5 mM Phenylmethanesulfonyl fluoride (PMSF, Merck) and 40 μl/ml “Complete Mix” protease inhibitor cocktail (Roche)]. The extracts were incubated with 40 μl agarose-conjugated anti-Flag antibody (M2, Sigma) at 4°C overnight. Precipitates were washed 6 to 8 times with CHAPS lysis buffer and finally resuspended in SDS-polyacrylamide gel loading buffer. For western blotting the proteins were resolved in SDS-polyacrylamide gels and transferred electrophoretically at room temperature to PVDF membranes (Merck) for 1 h at 50 mA using a Tris-glycine buffer system. The membranes were pre-blocked for 1 h in a solution of 3% milk powder in PBS-T (0.1% Tween 20 in PBS) before adding antibodies. The following antibodies were used: anti-GFP (7.1/13.1, mouse monoclonal IgG, secondary antibody peroxidase conjugated sheep anti-mouse IgG, NA931V, GE healthcare) or anti-Flag (M5, Sigma; secondary antibody, NA931V, GE healthcare).
Na931v
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden, Canada
The NA931V is a laboratory equipment product designed for general laboratory applications. It serves as a device for performing various analytical tasks and procedures. The core function of the NA931V is to provide users with a reliable and versatile tool for their laboratory needs.
Automatically generated - may contain errors
Lab products found in correlation
Na934v, by GE Healthcare (49 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Na934, by GE Healthcare (4 mentions)
Chemidoc imager, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Mab1501, by Merck Group (3 mentions)
Ab16048, by Abcam (3 mentions)
Western lightning ultra, by PerkinElmer (3 mentions)
Na935v, by GE Healthcare (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Na9340v, by GE Healthcare (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
A11017, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Ab114126, by Abcam (2 mentions)
Ab3280, by Abcam (2 mentions)
Ab4729, by Abcam (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
T6199, by Merck Group (2 mentions)
F7425, by Merck Group (2 mentions)
108 protocols using na931v
1
Immunoprecipitation and Western Blot Analysis
2
Immunoprecipitation and Western Blot Analysis
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HEK293 cells were transfected with the indicated constructs for expression of GFP- and Flag-tagged WT and mutant proteins. 24 hours after transfection cells were lysed with 600 μl CHAPS lysis buffer [10 mM 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, Roth), 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM NaF, 1 mM Dithiothreitol (DTT, Merck), 0.5 mM Phenylmethanesulfonyl fluoride (PMSF, Merck) and 40 μl/ml “Complete Mix” protease inhibitor cocktail (Roche)]. The extracts were incubated with 40 μl agarose-conjugated anti-Flag antibody (M2, Sigma) at 4°C overnight. Precipitates were washed 6 to 8 times with CHAPS lysis buffer and finally resuspended in SDS-polyacrylamide gel loading buffer. For western blotting the proteins were resolved in SDS-polyacrylamide gels and transferred electrophoretically at room temperature to PVDF membranes (Merck) for 1 h at 50 mA using a Tris-glycine buffer system. The membranes were pre-blocked for 1 h in a solution of 3% milk powder in PBS-T (0.1% Tween 20 in PBS) before adding antibodies. The following antibodies were used: anti-GFP (7.1/13.1, mouse monoclonal IgG, secondary antibody peroxidase conjugated sheep anti-mouse IgG, NA931V, GE healthcare) or anti-Flag (M5, Sigma; secondary antibody, NA931V, GE healthcare).
3
Western Blot Analysis of T Cells
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Western blot analysis was performed on the T cells cultured in vitro, as previously described [44 (link)]. The cells were washed with PBS, lysed with sodium dodecyl sulphate (SDS) sample buffer (2% SDS, 10% glycerol, 50 mM Tris-HCl (pH 6.8)), mixed with bromophenol blue and dithiothreitol (final concentration, 100 mM), and incubated at 95 °C for 5 min. The lysates (5 µg of protein) were then fractionated by SDS-polyacrylamide gel electrophoresis on a 5–20% gradient gel (Pagel; Atto, Tokyo, Japan), and the separated proteins were transferred to a nitrocellulose membrane (Hybond; GE Healthcare, Boston, MA, USA). The membrane was incubated with primary antibodies, including mouse anti-drebrin and mouse anti-β-actin (A5316, clone AC-74; MilliporeSigma, Burlington, MA, USA). The immune complexes were detected using HRP-conjugated anti-mouse secondary antibodies (NA931VS; GE Healthcare, Boston, MA, USA) and enhanced chemiluminescence reagents (ImmunoStar LD; Wako, Osaka, Japan). Images were obtained using ChemiDoc Touch (Bio-Rad, Hercules, CA, USA).
4
Antibody Validation for CHD8 Analysis
5
Western Blot Analysis of CHD8 Protein
6
Western Blot Analysis of Notch Signaling
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Western blot was performed on 15μg of cell lysates subjected to SDS-PAGE electrophoresis in an 8% gel and subsequently blotted to nitrocellulose membrane. Membranes were stained with primary antibodies anti-Notch3 at 1:1000 (Santa Cruz Biotechnology Inc., sc-5593), anti-Notch1 at 1:1000 (Cell Signaling #3608), or anti-α-Tubulin at 1:1000 (Sigma-Aldrich, T6074) and secondary peroxidase-conjugated antibodies goat anti-rabbit at 1:5000 (Sigma-Aldrich, A6154) and sheep anti-mouse at 1:5000 (GE Healthcare Life Sciences, NA931VS). Enhanced chemiluminescence was used to detect secondary antibodies (Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate, or GE Healthcare Life Sciences Amersham ECL).
7
CHD8 Antibody Western and ChIP Protocols
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Antibodies used in westerns and immunoprecipitation (IP)-westerns: anti-CHD8 N-terminal antibody (ab114126, Abcam), anti-CHD8 C-terminal antibody (11891S, Cell Signaling), rabbit purified IgG (3900S, Cell Signaling), anti-actin antibody (ab3280, Abcam), horseradish peroxidase (HRP)-conjugated donkey anti-rabbit secondary antibody (NA934, GE), HRP-conjugated donkey anti-mouse secondary antibody (NA931VS, GE). Antibodies used in ChIP: anti-CHD8 N-terminal antibody (ab114126, Abcam) and anti-H3K27ac (ab4729, Abcam).
8
Quantitative Western Blotting of Atxn1 KI Mice
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For Western blotting, brain tissues from mutant Atxn1 KI mice or littermate control mice were washed three times with ice-cold PBS and dissolved in lysis buffer containing 62.5 mM Tris–HCl pH 6.8, 2% (w/v) SDS, 2.5% (v/v) 2-mercaptoethanol and 5% (v/v) glycerol. Samples from cultured cells were prepared similarly. The protein concentration was quantified using the BCA method (Micro BCA Protein Assay Reagent Kit; Pierce Chemical, Rockford).
Primary and secondary antibodies were diluted for immunoblotting as follows: rabbit anti-HMGB1, 1:1,000 (ab18256, Abcam, Cambridge, UK); mouse anti-HMGB1, 1:1,000 (AHM0915, ATGen, Seongnam-si, Korea); rabbit anti-FLAG, 1:2,000 (F7425, Sigma, MI, USA); mouse anti-phospho-H2AX (γH2AX), 1:500 (Ser139, #05-636, Millipore, MA, USA); mouse anti-1C2, 1:1,000 (MAB1574, Millipore, MA, USA); rabbit anti-ubiquitin, 1:1,000 (Z0458, DAKO, Glostrup, Denmark); rabbit anti-HP1α, 1:1,000 (2623S, Millipore, MA, USA); rabbit anti-Cox IV, 1:1,000 (ab16056, Abcam, Cambridge, UK); mouse anti-α-tubulin, 1:1,000 (T6199, Sigma, MI, USA); mouse anti-GAPDH, 1:5,000 (MAB374, Millipore, MA, USA); and HRP-conjugated anti-mouse IgG and anti-rabbit IgG, 1:3,000 (NA931VS (mouse) and NA934VS (rabbit), GE Healthcare, NJ, USA). Antibodies were diluted in TBST with 5% skim milk.
Primary and secondary antibodies were diluted for immunoblotting as follows: rabbit anti-HMGB1, 1:1,000 (ab18256, Abcam, Cambridge, UK); mouse anti-HMGB1, 1:1,000 (AHM0915, ATGen, Seongnam-si, Korea); rabbit anti-FLAG, 1:2,000 (F7425, Sigma, MI, USA); mouse anti-phospho-H2AX (γH2AX), 1:500 (Ser139, #05-636, Millipore, MA, USA); mouse anti-1C2, 1:1,000 (MAB1574, Millipore, MA, USA); rabbit anti-ubiquitin, 1:1,000 (Z0458, DAKO, Glostrup, Denmark); rabbit anti-HP1α, 1:1,000 (2623S, Millipore, MA, USA); rabbit anti-Cox IV, 1:1,000 (ab16056, Abcam, Cambridge, UK); mouse anti-α-tubulin, 1:1,000 (T6199, Sigma, MI, USA); mouse anti-GAPDH, 1:5,000 (MAB374, Millipore, MA, USA); and HRP-conjugated anti-mouse IgG and anti-rabbit IgG, 1:3,000 (NA931VS (mouse) and NA934VS (rabbit), GE Healthcare, NJ, USA). Antibodies were diluted in TBST with 5% skim milk.
9
Quantifying PMS2 Expression by Western Blot
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SDS‐PAGE and western blots were performed as described previously (D'Arcy et al., 2019 (link)). Antibodies used for the western blots include: PMS2 mouse monoclonal antibody (1:1000; Santa Cruz Biotechnology, Cat # sc‐25,315) and MLH1 mouse monoclonal antibody (1:1000; Santa Cruz Biotechnology, Cat # sc‐133,228 X). For the loading control, PCNA, a (D3H8P) XP rabbit monoclonal antibody (1:1000; Cell Signaling, Cat #13110S) was used. An ECL anti‐mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000, GE Healthcare NA931VS, from sheep) or anti‐rabbit IgG secondary antibody conjugated to HRP (1:10,000, GE Healthcare NA934V, from donkey) and Advansta Inc Westernbright Sirius—femtogram substrate were used to visualize antibody binding using a Bio‐Rad ChemiDoc imager. As performed previously, relative PMS2 expression was quantified and normalized to PCNA using ImageLab 5.2.1 software (Bio‐Rad). The mean ± SEM was derived from the cumulative data generated from three separate experiments. Statistical analysis was performed in GraphPad Prism 7 using an unpaired t‐test with Welch's correction where equal standard deviations are not assumed.
10
Western Blot Analysis of Hep3B and U-937 Cells
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Hep3B cells were grown in 60 mm dishes, washed with PBS (2x), and collected by scraping in 500 μL RIPA buffer (10 mM Tris-CL, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM PMSF). U-937 cells were collected by centrifugation, washed with PBS, and resuspended in 500 μL RIPA buffer. Cells were mechanically lysed on ice using a syringe with a 27-gauge needle. Protein samples were mixed with an equal volume of 2x Laemmli SDS-sample buffer, placed in near boiling water for 5 minutes, and subjected to centrifugation at 15,000 x g for 10 minutes at 4 °C. Protein samples (25 μg) were separated using SDS-PAGE on a 10% polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-FLAG (1:1000; Millipore MAB3118, from mouse). ECL anti-mouse IgG secondary antibody conjugated to HRP (1:10,000 GE Healthcare NA931VS, from sheep) and SuperSignal West Femto Substrate were used for visualization.
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