Hello everyone, I conduct the western blot for the protein Leucine-rich repeat kinase 2 (LRRK2) in human brain samples. I used several antibodies such as ab133474 and # 73-253 etc, but the bands of LRRK2 are very weak. I think that maybe this problem is related with the conditions for WB. Could anymore who did WB for LRRK2 give me some details of the conditions that you used? Or the antibodies you used? Thanks so much.
Dear Xiaoye, I suggest titration of your antibodies: You could first try to increase the concentration of your secondary as much as possible (until background becomes detectable) and then increase the concentration of your primary as much possible. To save AB, starting with a dilute solution in a suitable buffer like PBST with a little sodium azide as preservative and blocking agent (BSA, NFDM etc) is fine and then adding more and more antibody. You later may dilute again if the AB conc. was too high (re-using AB dilutions works fine). Or prepare a set of AB dilutions and test with a set of blotting membranes and combine the solutions later and dilute them as appropriate.
If you are using digital image acquisition, rather look at significant differences between sample conditions and background than at absolute values.
Hello Xiaoye Sang ,
I would recommand you to read Dr. Mark Cookson's papers, who's mainly focus on LRRK2 interactions with the endo-lysosomal system and it's effect on the protein homeostasis in synucleinopathies.
He is used to do WB targetted against LRRK2 either in cells or in mice's brain.
Some of his paper benefits from Star Methods (thanks Cell Press!), which described quite well the procol and the antibody used like this one : Article The Parkinson’s Disease Protein LRRK2 Interacts with the GAR...
If you want to use the Li-Cor system (like in the paper linked here), I would be happy to explain you the step since you have one available at your institution.
Hope it helps,
Maxime.
Factors like concentration of the protein loaded, % of acrylamide gels used, rate of transfer onto either activated PVDF or nitrocellulose membranes, Ab concentration used and finally the exposure are some of the keys factors decide the fate of the final outcome. Given the size of LRRK2 (250kDa), acrylamide gels of 10% or less are suitable for resolution and transfer of LRRK2 protein as higher percentages impede complete LRRK2 transfer to membranes. Try protein transfer overnight at 35V at 4°C. Ab for LRRK2 from Abcam clone MJF2, cat # ab133474 (the one you tried) was already shown to work well by several authors. Check successful protein transfer with Ponceau S (Sigma) stain before staining the membrane with Ab. Given a good protein transfer, optimism the antibody concentration. Also, try primary antibody staining overnight at 4°C.
Most of the info. I suggested are very simple but play a key role for successful WB.
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