Anti-Haspin antibody (ab21686)

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Whole cell lysate of HEK293A, SHSY5Y, A549 treated with 50ng/ml of Nocodazole for 12 hours.

PRIMARY ANTIBODY Concentration or dilution 1:1000 to 1:100 Diluent buffer 5% Skim milk in TTBS Incubation time 12-20 hours Incubation temperature: 4oC

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED GAPDH was nicely visualized with all the same steps and materials stated above.

ANTIBODY STORAGE CONDITIONS -20C

SAMPLE PREPARATION After twice washings with PBS, cells were harvested and the lysed with lysis buffer (20mM Tris (pH 7.5), 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 10mM Na3VO4, 1µg/ml Leupeptin, 1ug/ml Aprotinin, 1mM PMSF). Reducing agent 5% beta-mercaptoethanol and 50mM DTT Boiling for ≥5 min? 5 minutes

AMOUNT OF PROTEIN LOADED Up to 50ug

ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE, 12%

TRANSFER AND BLOCKING CONDITIONS Lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane at 100V for 60 minutes. Transfer buffer is composed of 25 mM Tris, 192 mM glycine and 20% methanol. Membrane was blocked with TTBS (150mM NaCl, 10mM Tris-HCl (pH 7.4), 0.05% Tween 20) containing 5% skim milk for 30min, and incubated with selected primary antibodies O/N

Protein transfer verified -verified by Ponceau S Red staining

SECONDARY ANTIBODY - Goat anti-Rabbit/blocking solution/1:1000/16 hours at 4oC/washed threetimes in TTBS for 10min with gentle rotation.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Dilution ratio was concentrated up to 1:100 and loading amounts of cell lysates was increased up to 100ug.

ANSWER:

Thank you for contacting Abcam regarding ab21686.

I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the protocol information provided and would like to make some suggestions to improve the results with this antibody.

Have you tested the antibody with BSA as a blocking reagent? I know we have some data on our website to suggest that milk should also work, but we have tested and validated recent lots with 5% BSA as a blocking agent and antibody diluent. I would encourage you to test this as we have seen strong positive results with this protocol.

I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions or if these suggestions do not improve your results with this antibody.