Anti-Huntingtin antibody [1A771] (ab13583)

Further to previous correspondence >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Here is a powerpoint figure with the WB as I was explaining this matter. Even at lower dilutions than described in the ABCAM manual we could barely detect the positive control. I would like to point out as well that we used the identical positive control for both experimnets.

ANSWER:

Thank you for getting back to me with further details.

Once again I am sorry to hear of the difficulties that you have been experiencing with this antibody by western blotting. I am certainly prepared to offer you a credit note to the value of one vial of this antibody provided that the purchase was made within the past 90 days. If this is the case please e-mail details of the order including the order number and date of purchase and I will request that our accounts team process this request.

I look forward to hearing from you.

BATCH NUMBER 869995 ORDER NUMBER 53907

DESCRIPTION OF THE PROBLEM Our positive control is a 171 amino acids of huntingtin with 82Q overeprpressed in HEK293 probed with the polyglutamine antibody 1C2 (Chemicon), who showed us our fragment at 46 kDa overexpressed. We used the same sample as positive controls for the WB.

We diluted this antibody about as described to 1 ug/ml but saw nothing not even a positive control which was the first 171 amino acids of huntingtin with 82Q overeprpressed in HEK293. We decided to go up to 5 ug/ml after which we saw the hint of a band only the positive controls after 2 min which was ony slighly above backround.

SAMPLE Hek293 Hek293 cells +N171-82Q (positive control) Nontransgenic mouse brain extract Transgenic mouse brain extract N171-82Q

PRIMARY ANTIBODY Overnight in 5% Milk-PBS ab13583 Washing 3 x 10 min PBST

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Hek293 Hek293 cells + huntingtin N171-82Q (positive control) positive control which was the first 171 amino acids of huntingtin with 82Q overeprpressed in HEK293.

ANTIBODY STORAGE CONDITIONS as described in -20 C except we added up to 50% glycerol to avoid frezing and thawing.

SAMPLE PREPARATION HEK293 cells were diluted in PBS and PI, homogenized, transfered in 1x LSB, sonicated, heated and loaded on a 10 % SDS-gel.

AMOUNT OF PROTEIN LOADED 30 ug per lane

ELECTROPHORESIS/GEL CONDITIONS Reducing

TRANSFER AND BLOCKING CONDITIONS Transfer in1 x TG-SDS 8 hrs at 30 V coldroom, blocking 2-3 hr 5 % MILK-PBS We have found this Transfer buffer to be more efficient in tarnsfering poluglutamine proteins.

SECONDARY ANTIBODY PIERCE, Mouse, in 5% MILK-PBS, 1:25.000 for 1 hour as described in Manual of Pierce. Washing 3 x 10 min PBST followed by 2x PBS

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:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. Quality is important to Abcam.

You are using an ideal system for the testing of this antibody and have tried many of the suggestions that I would recommend including an overnight incubation of the antibody and an increase in the concentration of the antibody. However, I would appreciate it if you could e-mail me a representative blot image where you have successfully detected polyglutamine in your positive control.

I would also appreciate it if you could tell me why you are detecting your 171 residue positive control at 46KDa? Is it a consequence of having 82Q residues?

You also mention that you find that milk is the most appropriate transfer agent. Please can you tell me whether you have considered or tried using BSA as I would recommend this given the more specific and sensitive results obtained routinely.

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