His-tag | 3xHis (polyclonal)

KLH-conjugated synthetic peptide 3xHis

Samples:
40 µg of E. coli supernatant in control conditions at 3h (1)
40 µg of E. coli pellet in control conditions at 3h (2)
40 µg of E. coli supernatant induced at 3h (3)
40 µg of E. coli pellet induced at 3h (4)

40 µg of total protein from Escherichia coli expressing pGEX-PEX11a inducible by IPTG, extracted with HEPES 50 mM pH 7.6, 300 mM NaCl, 5% glycerol, 20 mM imidazole, 1 mM MgCl2, 1 x protease inhibitor; prepared in 0.063 M Tris-HCl buffer, pH 6.8, containing 2% sodium dodecyl sulfate (SDS; w/v), 10 % glycerol (v/v), 0.006 % bromophenol blue (w/v) and 10 mM DTT; and denaturated at 95 ºC for 5 min. Proteins were separated on 12 % SDS-PAGE and blotted 1 h to PVDF using semi-dry transfer. Blots were blocked with 3% (w/v) skimmed milk powder diluted in TBS-T O/N at 4 ºC with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5.000 for 1 h at RT with agitation in TBS-T + 3% (w/v) of skimmed milk powder. The antibody solution was decanted and the blot was washed 3 times for 10 min in TBS-T + 3 % (w/v) of skimmed milk powder at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, Product No: AS09 602) diluted to 1:10.000 in TBS-T + 3 % (w/v) of skimmed milk powder for 1 h at RT with agitation. The blot was washed as above but with TBS and developed for 5 min with chemiluminescent detection reagent following manufacture's recommendations. Exposure time was 30 seconds.

Courtesy Dr. uisa María Sandalio González, CSIC, Spain