Hi there, I've been trying to detect oligomeric bands of a protein (diseased mouse brain tissue) in western blot for some time. Immunostaining showed large aggregates of this protein in our diseased model's brains compared to none in control. However, I could only see a "monomer" band (14-16kDa) in both diseased and normal tissues. Does anyone know how to detect such kind of protein aggregates(oligomeric structures) in WB?
I used the lysis buffer which contains 20mM Tris,500mM NaCl and 2%SDS (is it too harsh for all oligomers to break into monomers?). I also added reducing agent and 5' boiling before loading into 4-12% bis/tris gel and run for 1 hr at 140V. Then transfer into PVDF membrane with 30V for 1 hr before processing further with 5% milk powder blocking and its antibody.
Any idea or suggestion is welcome. Thanks a lot!
You may try a native electrophoresis followed by WB, I attach here a protocol. Alternatively a good way to analyse aggregation state is to perform a gel filtration on FPLC. I hope to be of some help
You may try a native electrophoresis followed by WB, I attach here a protocol. Alternatively a good way to analyse aggregation state is to perform a gel filtration on FPLC. I hope to be of some help
Hello Dolors, I have found a paper which used crosslinkers like DSP, DSG, DSS to reveal the oligomeric structures of alpha-synuclein protein. I will have a look at PICUP method for further details. Thank you.
Hello Maria, I was also willing to test in a native gel as well. But I'm not sure if it might work because I have been told that the size of aggregates (beta-amyloid or alpha synuclein in my case) is too large to even pass through any kind of gel. Would that also be helpful with Thioflavin T or Congo red staining to see the aggregation in my samples? Thanks a lot.
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