Anti-HDAC6 antibody (ab12173)

LOT NUMBER GR1617-1 NUMBER 978213 DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE whole cell lysates human and mouse MEF cell PRIMARY ANTIBODY ab12173 1:1000 dilution, in TBST O/N at 4 C wash with TBST for 20 min three times DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Not used ANTIBODY STORAGE CONDITIONS -20 C SAMPLE PREPARATION 1% triton-X100 Lysis buffer/ Potease inhibitor: aprotinin/leupeptin/PMSF Phosphatase inhibitors: Na3VO4/NaF Reduvin reagent: DTT Heating for >5min AMOUNT OF PROTEIN LOADED 40 ug/lane ELECTROPHORESIS/GEL CONDITIONS 10 % reducing gel TRANSFER AND BLOCKING CONDITIONS PVDF membrane/ verified using Ponceau S Blockinb with 5% milk-TBST for 1hour at room temp SECONDARY ANTIBODY anti-Rabbit HRP 1:10000 in in TBST for 1 hr at room temp wash with TBST for 20 min three times HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? We changed amount of protein smaple for loading and incubation time with primary antibody.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us.

I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. 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 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund . I would like to reassure you that this antibody is covered by our guarantee in western blot, human and mouse.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab12173. I would also appreciate if you can confirm some further details:

1,  At what sizes were the non specific bands?  I would appreciate if you are able to provide an image, including molecular weight markers,  which would help us to asses the results.

2. Has the quality of the sample been assessed using a loading control?  

3. I can recommend to try a lower concentration of 1:2000 or even 1:3000 which may help to reduce the background.  

4. Could you confirm if the current vial of secondary antibody working well with other primary antibodies?  What were the results of a no primary control?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested information and details of how you would like to proceed.  

Customer would like to know the concentration of this antibody (he needs to know for conjugation purposes). Abcam lot# 102676.

ANSWER:

Thank you for your enquiry. The source of ab12173 has told me that they only have the protein concentration as determined by UV, for this IgG fraction. It is 5.9 mg/ml.

If you have any additional questions, please contact us again.