Immunoblotting assays were performed as described [33] (link). Primary antibodies were obtained from the following sources and used according to manufacturer’s recommendations: Cell Signaling Technology: anti-ERK (9102), phospho-eIF4E (9741), eIF4E (2067), P-4E-BP1 (9644), P-4E-BP1 (2855), anti-FRA (5281), phospho-FRA1 (5841), SPRY2 (14954), SOX2 (14962), MITF (12590), cyclin D1 (2978), P-ERK5 (3371), ERK5 (3372), p-p38 (4511), p38 (8690), p21 (2947), p27 (3686), EZH2 (5246), SUV39H1 (8729), H3K27me3 (9733), H3K9ac (9649), H3K27ac (8173), H3K4me3 (9751), H3 (4499), c-Met (8198); Abcam: histone H3.3-phospho S31 (ab92628), ATF3 (ab216569), SOX10 (ab216020), cystatin C (ab109508), SUV39H2 (ab190870); tubulin-HRP (21058). SOX17 (24903-1-AP); Thermo Fisher Scientific: SOX4 (PA5-40681); and Novus Biologicals: PML (NB100-59787). Covance: anti-HA monoclonal antibody HA.11. Secondary antibodies: Anti-mouse (7074) or rabbit IgG-HRP (7076) Cell Signaling.
Anti ha monoclonal antibody ha 11
Manufactured by Fortrea
The Anti-HA monoclonal antibody HA.11 is a laboratory reagent used to detect and identify proteins that have been tagged with the HA (hemagglutinin) epitope. It is a highly specific antibody that binds to the HA tag, allowing for the detection and analysis of HA-tagged proteins in various experimental applications.
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Lab products found in correlation
P 4ebp1, by Abcam (1 mentions)
H3k4me3, by Abcam (1 mentions)
Monoclonal anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
Anti rabbit irdye 680, by LI COR (1 mentions)
Anti mouse irdye 800, by LI COR (1 mentions)
Monoclonal anti gfp antibody, by Roche (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti ha monoclonal antibody ha 11
1
Immunoblotting Antibody Validation Protocol
2
Immunoblotting of HA-tagged Proteins
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Cells were lysed in RIPA buffer (20 mM Tris pH 7.4, 10% glycerol (137 mM), 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 200 μl of 1 mM PMSF and protease inhibitor cocktail). Equal amounts of protein (30 μg/lane; Bradford method) were electrophoresed in 15% SDS polyacrylamide gel. Blocked membranes (2% BSA in 10 mM Tris-buffered saline, 0.05% Tween (TBST) for three hours at 24 °C) were incubated overnight at 4 °C with anti-HA monoclonal antibody (HA.11, 1:1000; Covance, Inc., Berkley, CA). Membranes were washed three times with TBST and incubated with goat-anti-mouse horseradish peroxidase-labelled antibody.
3
Antibody Characterization for S. cerevisiae Proteome
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S. cerevisiae strains used in this study are described in Table S1. Anti-HA monoclonal antibody (HA.11, raised in mouse) and anti-myc monoclonal antibody (9E10 c-myc, raised in mouse) were purchased from Covance. Anti-Kar2 and anti-Sec61 antibodies (rabbit) were provided by P. Walter (University of California, San Francisco, San Francisco, CA). Anti-Sis1 antiserum (rabbit) was a gift from D. Cyr (University of North Carolina, Chapel Hill, NC). Monoclonal antiproteasome 20S α (mouse) and polyclonal anti–histone H3 (rabbit) were purchased from Abcam. Monoclonal anti–3-phosphoglycerate kinase antibody (mouse) and monoclonal anti-V5 antibody (mouse) were purchased from Invitrogen, monoclonal anti-Ydj1 antibody (mouse) was from StressMarq, monoclonal anti-GFP antibody (mouse) was purchased from Roche, and monoclonal anti-FLAG (mouse) was purchased from Sigma-Aldrich. Secondary antibodies labeled with Alexa Fluor 488 (anti–mouse) or Alexa Fluor 596 (anti–rabbit) were purchased from Molecular Probes. Anti–rabbit IRDye 680 and anti–mouse IRDye 800 secondary antibodies were purchased from LI-COR Biosciences. Anti-Ubr1 antiserum was raised in rabbit against a recombinant protein containing the 100 N-terminal amino acids of Ubr1. Anti-San1 antiserum was raised in rabbit against a protein containing the 100 C-terminal amino acids of San1.
4
CRF1R-EGFR-Src Co-immunoprecipitation
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COS-7 cells transfected with HA-CRF1R were grown in 10-cm dishes and serum-deprived for 24 h before treatment with 100 nM CRF for 10 min at 37°C. Cells were washed twice with ice-cold PBS and lysed in Nonidet P-40 solubilization buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM Orthovanadate, 1 mM NaF, 1% Nonidet P-40, 10% Glycerol, 2 mM EDTA, pH 7.4, containing protease inhibitors). After immunoprecipitation of HA-CRF1Rs with anti-HA monoclonal antibody (HA.11; Covance, San Diego, CA) and protein A/G PLUS-Agarose (Santa Cruz Biotechnology, CA), the proteins were resolved by SDS-PAGE, Western blotted, and probed with anti-EGFR polyclonal or anti-HA monoclonal antibodies, followed by a horseradish peroxidase conjugate to identify co-immunoprecipitated proteins. Blots were also stripped with stripping buffer (100 nM Glycine-HCl, pH 2.7) and reprobed with anti-c-Src polyclonal antibody. Western blot detection of co-immunoprecipitated Src was carried out as described above. Blots were visualized and quantified, as indicated above.
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