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Please find attached the western blot performed with ab75054 , that worked. This was done using 30ugs total cell lysates, o/n incubation of antibody at 1:500 in 5% milk/0.1%Tween.
Thanks for working with me on this!
Asked on Oct 03 2012
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
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
- 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.
Answered on Oct 03 2012