Anti-RAB14 antibody (ab40938)

Dear Abcam,

I recently bought the Rab14 antibody (ab40938) and apparently -in my hands- it is not working for Western blot (WB)and IP followed by WB (see attachments).

Regarding attachment "RAB14 (ABCAM)...."this WBwas loaded with mousemuscle homogenates of 10, 30or 60 ug total protein per wellas well as a positive control in the form ofC2C12 muscle cells overexpressing Rab14homogenates (12 ug protein per well)and AB concentrations of 1:5,000 (left membrane)or 1:10,000 (right membrane)using ECL and CCD detection.

Regarding attachment "Rab14 IP..." I see bands at 25 kDain the four first rows fromthe left after the marker lane(which are the loaded IPs with 10, 7.5, 5 and 2.5 ug AB) as well asone 50 kDa bandin the first row from the left after themarker. I presume these are just light and heavy chains. Importantly, in the lane after these four rows with 25kDa bands, I loaded a positive control (C2C12 muscle cells overexpressing Rab14homogenates (12 ug protein per well) and I see no signal at all. The WB was done with and AB conc.at 1:5,000.The following 4 last lanes are the corresponding post-IPs.

Could you please give me your input to this. Is this AB not working properly or am I doing something wrong?

Thank you.

ANSWER:

Thank you for your email.

I am sorry to hear you have been experiencing problems with our Rab14 antibody. The quality of our products is important to us so thank you for contacting us with more information. I have some further questions and also some suggestions on how to improve the results:

- It is possible to obtain the batch number of the vial received? This will help us investigate the case further.
- Can you confirm that Rab14 is expressed in C2C12 cells? I couldn't find any reference showing this.
- We use 3% BSA for blocking and antibody incubations. If you have used milk it would be worth switching blocking agents as this might help.
- Rab14 should be observed in the overexpressing homogenates. How did you confirm that the protein is being expressed. Is the construct tagged with a marker?
- Rab14 is ˜25 kDa so the target protein may not be observed due to the reaction with light chain in IP. I would suggest cross-linking the antibody to the beads. Here is a link to a protocol:

http://www.abcam.com/index.html?pageconfig=resource&rid=11415

Please let me know how you get on. If you are still experiencing problems and the product was purchased in the past 6 months. I would be happy to offer a replacement or refund.