Immunofluorescence - NFkB p50 antibody (ab7971)

HeLa cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/500 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted. Red indicates staining by ab7971, blue staining by DAPI.

Michael Mancini, Baylor College of Medicine

ChIP - NFkB p105 / p50 antibody - ChIP Grade (ab7971)

X-ChIP with ab7971 showing enrichment of NFkB p105 / p50 at regions upstream of transcription start site of the NFKBIA and IL8 genes. Chromatin was extracted from monocytes treated with LPS and the proteins were cross-linked to DNA with formaldehyde for 15 minutes. The immunoprecipitation step involved incubation of the cross-linked chromatin with ab7971 for 16 hours at 4ºC. Please see abreview by Genpathway for additional details.

This image is courtesy of an Abreview submitted by Genpathway Inc

Immunocytochemistry/ Immunofluorescence - NFkB p105 / p50 antibody - ChIP Grade (ab7971)

ICC/IF image of ab7971 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7971, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Flow Cytometry - Anti-NFkB p105 / p50 antibody - ChIP Grade (ab7971)

ab7971 staining NFkB p105 / p50 in Human Jurkat cells by Flow Cytometry. Cells were prepared in a phosphate buffered solution containing 0.1% sodium azide with FBS, fixed with paraformaldehyde and permeabilized with Triton X-100 and NP40. The sample was incubated with the primary antibody (1/100 in wash buffer) for 24 hours at 4ºC. A FITC-conjugated Goat anti-rabbit Ig (1/100) was used as the secondary antibody.

Gating Strategy: Isolate cell population from plot of SSC-A / FSA-A

This image is courtesy of an anonymous Abreview