Anti-RNF144A抗体(ab75054)

Thanks for your reply with the images.

I was able to view the longer exposure image but the not the shorter exposure, I think the file was corrupted. However, I still got a good idea of the results with the long exposure. Here are my suggestions:

- Incubate the membrane at room temperature with less antibody (1:2,000 dilution). The background is high, so it is worth titrating the concentration to increase specific binding but reduced background.
- Block the membrane with at at least 1% milk, I wouldn't decrease this any further.
- Increase the washing time and perhaps the level of detergent? If you use Tween, you could switch to Triton (0.1%).
- Has the secondary antibody worked well with other primary antibodies? This will ensure the reactivity/sensitivity of the secondary has not changed.
- For sample preparation I would suggest using a RIPA based buffer or cellular fractionation in order to efficiently isolate the membrane

RIPA buffer
50 mM Tris HCl pH8
150 mM NaCl
1 % NP-40
0.5 % sodium deoxycholate
0.1 % SDS
2 mM EDTA
+ protease inhibitors

Here is a link to our subcellular fractionation protocol on our website
https://www.abcam.com/index.html?pageconfig=resource&rid=11473

- The lab confirmed that the antibody should be stored in single use aliquots.

I hope some of these improvements help and please let me know about your results.

Thank you for speaking with me today.

I have issued a free of charge replacement for ab75054 which is scheduled for dispatch today and should be with you tomorrow. Please aliquot the product into single use aliquots to prevent freeze thawing between each use. Once I receive the images from you I will consider what further changes to the protocol might help.

Looking forward to your reply with further information.

Thank you for your reply.

I would be happy to speak with you further about these antibodies. Just let me know when would be a convenient time for you.