I am treating neuroblastoma cells with AB42 protein (monomers and fibrils in separate experiments) and studying what happens after its uptake and concurrent interactions with intracellular exosomal machinery. Now, I plan to treat the cells for 24 hours, wash them with PBS and replace it with media without AB42 and incubating for another 48 hours.
In this time period I want to see if there is something happening to the AB42 inside the cell. For this I want to do a Western blot for the protein by preparing a cell lysate.
Are there any recommendations for a decent lysis buffer to use and whether or not I should use protease inhibitors? What are the best conditions for the Western Blot? I usually do it in a 4-12% Bis-Tris gel in 1X MES Buffer.
PS: I do not want to alter the structure of my protein for the western blot.
Thank you
Hi
You should first find theoretical pI of your protein and chose accordingly a buffer system(and pH) regarding buffering region pK +/- 1 and pI in this range. Additionally, you should include salts in your buffer depending on physiological ionic strength (between 100 – 200 mM KCl or NaCl). Protease inhibitors or protease inhibitor cocktails are essential.
I am not sure if I understand your question correctly. Do you want to do western blot or dot blot? because conventional western blot is a denaturing method, which will certainly alter your protein structure. Native PAGE and western blot might be helpful, however the structure will be altered in blotting step due to SDS in transfer buffer.
Best
Erman Kocak Thank you for the answer. I have looked into the theoretical PI of Abeta 42. It is at pH 5.5. I will look into appropriate buffers for this.
I want to do a western blot. I usually do it under non reducing conditions; i.e, with sample and 1/4th amount of LDS and load into my gels for SDS. I assume that does not alter my protein structure that much. I want to do a western blot after that to see if abeta in my cells is in monomeric of fibrillar form after treatment.
PS: I am sort of new to Western Blotting, so please be gentle. :)
Thank you
Hi Nandan
I wonder if you solved your problem and was possible for you to detect amyloid beta 42 by WB because I am currently trying to detect this protein with no success from an astrocytic lysate (cells transfected for APP). I know that the levels of amyloid beta can be really small, that´s why that I am trying to concentrate them.
Would be incredible helpful if you could give me any advice for the western blot protocol of this protein.
Thanks in advance
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