Anti-SIG5 | RNA polymerase sigma factor sigE

Samples:

1. MW standards
2. 6xHis-GST-AtmSIG6 (2ug eluate)
3. 6xHis-GST-AtmSIG5 (2ug eluate)
4. 6xHis-GST-AtmSIG5 (1ug eluate)
5. 6xHis-GST-AtmSIG5 (0.5ug eluate)
6. 6xHis-GST-AtmSIG5 (0.25ug eluate)
7. 6xHis-GST-AtmSIG5 (0.125ug eluate)
8. 6xHis-GST-AtmSIG5 (0.0625ug eluate)
9. 6xHis-GST-AtmSIG5 (0.03125ug eluate)

SIG5 was overexpressed in E.coli and varoius amounts of E.coli extract were loaded/well. 

Protein extracts were warmed for 5 min at 80ºC in thermo block. Debris was eliminated by centrifugation at 10,000 x G for 10 min, and supernatant was transferred to clean 1.7 mL tube for use. Concentration of total protein was determined via Pierce™ Detergent Compatible Bradford Assay Kit. A 2x dilution series, starting at 10µg of total protein separated via 10% acrylamide SDS-PAGE. Proteins were transferred to PVDF membranes for 30 minutes using Bio-Rad Trans-Blot Turbo Transfer System. Blot was blocked in 5% non-fat milk in TBS-T overnight at 4C with agitation. Blot was incubated with Agrisera anti-Sig5 primary antibody (AS23 4949) at a dilution of 1:1000 for 1hr at RT with agitation in TBS-T. The antibody solution was decanted, and the blot was rinsed briefly, then washed 3 times for 5 minutes in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG HRP conjugated, AS09 602) diluted to 1:25 000 in TBS-T for 1 hr at RT with agitation. The blot was washed as above and developed chemiluminescent detection reagent. The blot was then exposed for 0.1s, 1s, or 60s via Bio-Rad ChemiDoc MP imager. The blot was then imaged colorimetrically and merged with the 1s exposure to determine molecular weight using Bio-Rad Precision Plus Protein^TM All Blue Prestained Protein Standards. The blot was then stained with 0.1% Coomassie Brilliant Blue to account for proper protein transfer and loading.

Courtesy of Dr. Martin Matthew Ross, Department of Biochemistry and Center for Plant Biology, Purdue University, USA