Anti-CaMKII alpha (phospho T286) antibody (ab5683)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 927009

DESCRIPTION OF THE PROBLEM no bands

SAMPLE Rat hippocampus

PRIMARY ANTIBODY Manufacturer: Abcam Inc. Species: Rabbit Diluent: 5% milk made in tTBS Dilution: tried 1:1000, 1:500, and 1:250 Incubation time: 1 hour Wash step: washed 3 times for 15 minutes in tTBS

DETECTION METHOD Amersham ECL Western Blotting Detection Reagents (GE Healthcare)

POSITIVE AND NEGATIVE CONTROLS USED After we tried to probe membranes with this antibody we reprobed them with three other antibodies and got strong signals.

ANTIBODY STORAGE CONDITIONS The antibody was stored at -20 C and aliquoted after the first use.

SAMPLE PREPARATION Buffer: Homogenization buffer: 600ul 5M NaCl (150mM) 400ul 1M Tris pH 7.5 (20mM) 80ul 0.5M EDTA (2mM) 200ul 0.1M DTT (1mM) 200ul Triton (1%) 40ul CLAP 18.48ml H2O

Protease inhibitors: CLAP: Chymostatin Leupeptin Antipain Pepstatin

Heating: 5min at 95 C before loading

AMOUNT OF PROTEIN LOADED About 52ug. After we tried to probe membranes with this antibody we reprobed them with three other antibodies and got strong signals.

ELECTROPHORESIS/GEL CONDITIONS Non-reducing, 10% Tris/Tricine Polyacrylamide Gel

TRANSFER AND BLOCKING CONDITIONS Transfer buffer: 500ml 1X Tris/Tricine/SDS Running buffer 200ml Methanol 300ml dH2O

Transfer time period: 50 minutes

Blocking agent: 5% milk in tTBS for 1 hour

SECONDARY ANTIBODY Manufacturer: Jackson Immuno Research Species: Goat Anti-Rabbit IgG Horseradish peroxidase Diluent: 5% milk made in tTBS Dilution: tried 1:10,000 Incubation time: 1 hour Wash step: washed 3 times for 15 minutes in tTBS

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

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab5683 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a credit note/refund will be offered.

From the protocols questionnaire, I noticed that you used 5% milk as a blocking reagent and also as diluents for the primary and secondary antibodies. I believe that the reason why you are not seeing any bands is because cross reaction between the blocking agent and antibodies might have occurred. Here at Abcam, we recommend using 5% BSA as almost all of our products have been tested with it. Please try BSA as a substitute for milk.

One other possible reason why no bands were observed could also be due to insufficient antibody binding to protein of interest. A longer incubation time will ensure proper and sufficient binding. Please try incubating the primary antibody at 4oC, overnight if you have not already done so.

Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies?

I hope the above suggestions might prove to be useful in resolving the matter. Please try them out and should you still not get any results, please get back to me with your purchase details.

can this be used as a positive control in western blotting analysis of CaMKII alpha phosphorylated and non phosphorylated (ab5683 and ab3908 respectively).

ANSWER:

Thank you for your enquiry.

Yes, ab7942 (rat brain lysate) or ab7188 (mouse brain lysate) could be used as a positive control for ab3908 and ab5683. The previous email that I sent about ab10772 [CaMKII alpha peptide - phospho T286 (phospho and non-phospho pair)] also relates to these antibodies. This peptide pair can also be used to verify that the band you see is specific.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information,