Anti-SQSTM1 / p62 antibody (ab91526)

Thank you very much for your call today and for letting us know about the trouble with ab91526.

As we discussed, please keep me updated about the results after reducing the protein loading to 20, 40, and 60 ug per lane. If the results don't improve I'll be happy to send a replacement or issue a credit or refund.

I look forward to hearing from you. If you have any questions or need anything else, let me know and I'll be happy to help. Have a nice day!

Thank you for your inquiry.
If the cap of the vial has not been screwed tightly back again, it is very well possible that the remaining 80ul have been evaporated during the 3 weeks. Please recommend the customer to tightly screw the cap on the vials when storing them. In cases of doubt, the customer can also use parafilm to avoid any evaporation. This is however not necessary in general.

Thank you for providing that information.

Having reviewed the protocol used, I believe there are afew things that maybe worthtrying in order toimprove the specificity observed.I would suggest using a reducing agent in the loading buffer to reduce the aggregation which may be occurring (especially with ab91526). Either DTT or b-mecaptoethanol. It would also be worth reducing the temperature at which the protein is denatured. Certain proteins are prone to aggregation, which can be reduced by heating to 70 degrees for 10 minutes instead of 95. I would also suggest reducing the concentration used slightly (ab53255 down to 1/500 and ab91526 to 1/1000) to see if this improves the binding as well as reducing the amount of protein loaded (down to 40 ug/lane).

Has a "no primary" control been performed? This may be worthwhile, just to rule out any non-specificity relating to the secondary antibody used.

Should these suggestions not improve the results please do let me know and I'll look into if we have any alternative lots of these antibodiesor different antibodies altogetherwhich you may be interested in trying.

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewedthe information you have provided, I would like to confirm a few further details in order to understand the procedure used more fully and hopefully provide more specific protocol tips:

1. Do you know the lot number and order number of each of the antibodies?

2. How was the sample collected and treated prior to loading on the gel? What lysis buffer was used? Were any protease inhibitors used? Was the sample boiled prior to loading? Was any reducing agent used?

3. How was the blocking performed? Time/temperature? Was any detergent included in the blocking buffer?

4. How was the primary antibody incubated with the membrane (time/temperature)? What diluent was used with each of the antibodies?

5. What washing steps were employed and with what buffer?

6. Were the membranes freshly probed or was the same membrane used and stripped between each antibody? If so, how was this procedure performed?

Hopefully with this information I will be ableto suggest modificationswhich will improve the specificity currently observed.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I look forward to receiving your reply.

Thank you for contacting us.

There are number of reasons which may affect the apparent band size from one western blot to the next. In this particular case those may include the fact that this protein undergoes a number of post translational modifications which may greatly affect its size. The Abreview used a different species than the datasheet. The image in the Abreview was created by a customer and as such we do not have the exact protocol that they used including but not limited to gels and protein markers used and sample treatment.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.