Anti-ABCA1 antibody [HJ1] (ab66217)

AbID: 66217, Anti-ABCA1 antibody [HJ1] Rating: Below Average Image: Anti-ABCA1 antibody [HJ1] for Western blot (Human) Sample: Species: Human Type: Cell lysate - other Loading amount: 25 µg Specification: Mononuclear Cell Preparations Application: Application: Western blot Gel Running Conditions: Reduced Denaturing (4-15%) Blocking step: Milk as blocking agent for 1 hour at 22°C Blocking Concentration: 5% Other detail: Dilution: 1/1000 Incubation time: 16 hours at 4°C Diluent: 5% BSA in PBST Secondary Antibody: Name: Non-Abcam Antibody was used: Donkey anti-mouse HRP Conjugation: HRP Dilution: 1/10000 Detection: Detection method: ECL+ Exposure: 5 minutes and 0 seconds Bands: Specific: No kDa Non-specific: 48, 100, 120 kDa Additional Data:

ANSWER:

Thank you for submitting your recent review of ab66217. We are sorry to see that the results were unsatisfactory. I'd like to follow up with you to offer a couple of suggestions that may help improve these results. However we do fully guarantee this antibody to work in tested species and applications, so I would be happy to replace this antibody or issue a credit or refund if you would prefer. Some lower molecular weight bands are often seen with ABCA1 antibodies, and though the identity is not known, it is possible that these are cleavage fragments. Since the molecular weight of the full length ABCA1 is around 254 kDa, and this protein is heavily glycosylated, it might be difficult to transfer to the membrane. Did you do a Ponceau stain to verify the transfer? There are a few protocol alterations that we recommend when transfering such a large protein: 1) Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. We recommend 10% with nitrocellulose or none if using a PVDF membrane. 2) Choose wet transfer overnight at 4°C instead of semi-dry transfer. I look forward to hearing from you. If you would like a replacement, credit, or refund, please send me your order number and I will be happy to arrange this for you.