Anti-HNF4 antibody [K9218] - ChIP Grade (ab41898)

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Hepatocytes isolated from Lewis rats

PRIMARY ANTIBODY ab41898 - Mouse monoclonal to HNF4 25 minutes, 4°C

DETECTION METHOD flow cytometry

POSITIVE AND NEGATIVE CONTROLS USED negative control: only secondary antibody positive control: hepatocytes should be a positive control

ANTIBODY STORAGE CONDITIONS 4°C

SAMPLE PREPARATION Cells resuspended in DMEM buffer with foetal calf serum, penicillin/streptomycin and fungizone.

NUMBER OF CELLS USED 1 million cells

PERMEABILIZATION STEP No

BLOCKING CONDITIONS No

SECONDARY ANTIBODY goat anti-mouse IgG PE 15 minutes, 4°C

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

We recently carried out Flow cytometry and updated the datasheets in December.

Please Aliquot and store at -20°C long term. Avoid repeated freeze / thaw cycles. Storing the antibody at 4deg for extended period (more than two weeks) could affect the activity of the antibody.

I have looked through your protocol and would like to ask you some questions in order to understand your procedure better as well as make a few suggestions to improve your results: 1. How much antibody did you use? 2. Have you tried any optimization of the protocol?

If you scroll down on the online datasheet you will see the Flow cytometry testing image for HepG2 cells.

Conditions used were: Overlay histogram showing HepG2 cells stained with ab41898 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab41898, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a significantly decreased signal in HepG2 cells fixed with 4%[araformaldehyde/ permeabilized in 0.1% PBS-Tween used under the same conditions.

You need to fix your samples either with MeOH or PFA. Otherwise as the target is nuclear the antibody is not able to enter the cell. HNF4 is not on the plasmamembrane/extracellular, thus without sample fixation you will not detect it.