Whole cell lysates were performed using either IP buffer (50 mM Tris–HCl pH 7,5; 150mM NaCl; 1% NP-40; 5 mM EDTA; 5 mM EGTA; protease inhibitors) or Laemmli Buffer 4×. Protein extracts were resolved on SDS-PAGE polyacrylamide gels and transferred to nitrocellulose blotting membrane (GE Healthcare Life Sciences). Membranes were incubated with the following primary antibodies: α-FLAG M2 (Sigma-Aldrich, #F1804), α-STREP-HRP (Sigma-Aldrich, #RABHRP3), α-Vinculin (Sigma-Aldrich, #V9131), α-BirA (Abcam, #ab232732), α-WDR5 (Abcam, #ab56919). Secondary antibodies conjugated to horseradish peroxidase were used at 1:10 000 dilutions (Jackson Immunoresearch, anti-mouse IgG HRP #715–035-150, anti-rabbit IgG HRP #711-035-152). Signal was visualized using the ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, MA).
α bira
Manufactured by Abcam
Sourced in United Kingdom
α-BirA is a recombinant protein that catalyzes the biotinylation of target proteins. It is a useful tool for the site-specific labeling of proteins with biotin.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)
Anti rabbit igg hrp, by Jackson ImmunoResearch (1 mentions)
α flag m2, by Merck Group (1 mentions)
α vinculin, by Merck Group (1 mentions)
Anti mouse igg hrp, by Jackson ImmunoResearch (1 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
α flag m2 affinity gel, by Merck Group (1 mentions)
α flag, by Merck Group (1 mentions)
α β actin, by Merck Group (1 mentions)
Gfp trap resin, by Proteintech (1 mentions)
α abc, by Cell Signaling Technology (1 mentions)
α β actin, by Abcam (1 mentions)
α gfp, by Merck Group (1 mentions)
2 protocols using α bira
1
Western Blot Analysis of Protein Complexes
2
Plasmids, Antibodies, and Resins for Wnt Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The following plasmids have been described: LDB1-FLAG, SSDP-FLAG (Fiedler et al., 2015 (link)); murine FLAG-B9L, FLAG-ΔC (called FLAG-ΔCter), GFP-B9L (Adachi et al., 2004 (link)); GFP-Lgs, GFP-TCF4 (Townsley et al., 2004 (link)). Human GFP-BCL9 was generated by exchanging the FLAG tag of FLAG-BCL9 (de la Roche et al., 2008 (link)). Mutants were made from parental plasmids using standard site-directed mutagenesis procedures, and confirmed by sequencing. TLE3 was amplified by PCR from 6xMyc-TLE3 (Hanson et al., 2012 (link)) and subcloned in pcDNA3.1-N-HA, and mutants (WD40, ΔWD40) were generated from this subclone.
The following antibodies and resins were used: α-GFP RRID:AB_439690 , α-FLAG RRID:AB_262044 , α-HA RRID:AB_390918 , α-β-actin (for human lysates) RRID:AB_476744 (Sigma-Aldrich, St. Louis MO, USA); α-BCL9 RRID:AB_2063609 , α-BCL9-2 RRID:AB_2063747 (Abingdon, UK); α-β-actin (for fly lysates) RRID:AB_2305186 , α-BirA RRID:AB_300830 , α-Pygo2 RRID:AB_10863482 (Abcam, Cambridge, UK); α-ABC RRID:AB_11127203 (Cell Signaling Technology). Rat α-Lgs antiserum (Eurogentec, Liège, Belgium) was obtained from pre-bleed immunizations with gluthathione S-transferase-purified Lgs232-555. GFP-Trap resin RRID:AB_2631357 (Chromotek, Planegg, Germany) was used for coIP assays, and α-FLAG M2 affinity gel RRID:AB_10063035 (Sigma-Aldrich) was used for RIME.
The following antibodies and resins were used: α-GFP RRID:
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