Anti-Phospholipase C gamma 1 (phospho Y783) antibody (ab4828)

DESCRIPTION OF THE PROBLEM weak signals, therefore very low signal-background ratio

SAMPLE rat, endothelial cells from heart, whole cell lysate

PRIMARY ANTIBODY TBS containing 0.1% Tween, 5% BSA overnight at 4°C with ab4828 1:1000, 1:500, 1:200

DETECTION METHOD ECL Plus

POSITIVE AND NEGATIVE CONTROLS USED As positive controls: 2h 100ng PMA and 100µM ATP for 2 min not a real negative control: unstimulated cells

ANTIBODY STORAGE CONDITIONS aliquot, -24°C

SAMPLE PREPARATION Cell lysis: PBS w/o calcium, w/o magnesium; 1% NP-40; 1mM PMSF, 1µg/ml Pepstatin, 40mM beta-Glycerophosphate, Sigma Phosphatase Cocktail 2 mechanical destruction and sonification at 4°C Sample Preparation according to invitrogen/Nupage gels LDS Sample buffer, reducing agent, 10min 70°C heating

AMOUNT OF PROTEIN LOADED approx 150µg/band

ELECTROPHORESIS/GEL CONDITIONS NuPage Electrophoresis System, 4-12% Bis-Tris Mini-Gel, MOPS buffer, reducing agent, 200V constant 50 min

TRANSFER AND BLOCKING CONDITIONS PVDF membrane, Tansferbuffer Invitrogen, 1h 40 min 20V Ponceau TBS containing 0.1% Tween, 5% non-fat dry milk for 1 h 2x2min washing in TBS containing 0.1% Tween

SECONDARY ANTIBODY TBS containing 0.1% Tween, 5% non-fat dry milk for 1 h (other company)ECL anti-rab IgG (NA934V) 1:4000

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? primary antibody concentration (up to 1:200) and protein loading (maximum for these commercial gels)

ADDITIONAL NOTES I could detect PLC phosphorylation at tyr 771 with the identical protocol (good signal to background level). But just get really weak signals just below background with the tyr 783, and I only get these signals when I increase the detection time up to 20 min (!). (Biorad CCD detection system) I would like to know if there is a treatment/stimulation for my endothelial cells which causes a phosphorylation at tyr 783 without any doubt.

ANSWER:

Thank you for your enquiry. I am sorry to hear you are having a problem with ab4828 (Phospholipase C gamma 1 (phospho Y783) antibody).

The following information from the datasheet should help improve your results:

Extracts prepared from NIH-3T3 cells exposed to PDGF (10 ng/mL) for 10 minutes were resolved on a 10% Tris-glycine gel and transferred to nitrocellulose. Membranes were blocked with 1% BSA, followed by incubation with 0.5 µg/mL ab4828 antibody. After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase and the signal was detected by chemiluminescence using the Tropix WesternStar detection method and Kodak BioMax ultraclear film. The data show that PDGF is a strong inducer of PLC gamma 1 phosphorylation on Y783 in NIH3T3 cells.

The WB image is available on the ab4828 datasheet.

Please let me know if the above suggestions improve your results. Note that it is important to include the NIH-3T3 cells as a positive control.

I look forward to your reply.

Customer would like to know the concentration.

ANSWER:

The originator of ab4828 was able to give me a concentration for this antibody - 0.25 mg/mL. Please let me know if you have any more questions.

Customer would like to know the concentration.

ANSWER:

Thank you for your enquiry. There isn't a concentration for this glycerol format antibody. Ab4828 was packaged based on volume in order to be able to adjust the concentration of the antibody to assure consistent batch-to-batch signal strength using the originator's prequalified test systems. Therefore the actual amount of antibody is less critical than the signal intensity provided. For Western blotting it is recommended to use ab4828 at a 1:1000 dilution. If you have any more questions please contact us again.