Hi everyone,
I am a first year PhD student working on amyloidosis. I would like some tips and tricks to crack efficiently my current nightmare, i.e. the western blotting of the Amyloid beta and its oligomers. I am using brain endothelial cells to understand its presence in different oxidation condition, but as you might understand I have some problem with the WB technique (I am very naive about it).
I am using 16% tris-tricine gels, but my 2X Bio-Rad loading buffer contains Coomassie so, when i transferred my proteins to a 0.22 um NC membrane it stained horribly all the membrane, making impossible the detection of the amyloid-beta antibody with any detection method. Then another problem I got is that, since I need to load huge amount of proteins (around 60 ug), I had to overload my wells and the electrophoresis was horrible. All my bands were so thick and not sharp as they should be (or I am used to with tris-glycine gels).
So, do you have any advice on how to run effectively this kind of protocol? Also, any advice regarding how to obtain more concentrated lysate would be beneficial. At the moment my cell lysate has a concentration of around 2.80 ug/ul.
Ps: I am using a cerebral cortex human brain lysate as a positive control, together with synthetic AB40.
Thanks!
Hi Marco Antonazzi
Your protein concentration is not bad; I think it should work. Please follow the protocol exactly as described in the attached file. You may use the URL therein to watch the method video.
All the best!
Anoop
Hi Anoop Arunagiri
Thank you for your reply! Yes, I actually used that and I ha realised that if I was using an ECL-based detection method, which is an enzymatic reaction, my coomassie stained on the membrane wouldn't come up. I tried that, and actually it was as predicted.
Thank you so much again!
All the best,
Marco
The best way is to use an amyloid-beta kit, which is supplied by some specific suppliers.
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