I want to run the 18% gel so please tell me what should be the condition for that staring from running voltage upto transfer on membrane.
For isolation of protein from drosophila head for western for amyloid beta, which buffer is best?
Is there any suitable protocol for isolation of soluble and insoluble fraction of amyloid beta from drosophila head?
Dear Sandeep,
there are a few protocols out there for protein extraction. Have a look at the following for a start and do some more searches:
http://link.springer.com/content/pdf/10.1007%2F978-1-59745-257-1_27.pdf
The running and western conditions you need to use depend on what your set up is. Ie using mini gels, mode of transfer etc. I do not think that you need anything special so you can adopt a standard western procedure available on the net. Just make sure that the membrane size for transfer is correct for your protein size.
Cheers
about the western, probably you should use a tris-ticine gel, (a kind of "gradient" gel), wich is useful to separate small proteins, like a betta. I send to you a protocol with a brief explanation, also with the recipes, I think it is useful. you should start with 30 V until sample has enter stackin gel, and increase up to 120 V.
about the transference, because a betta is a small protein, you should use a transfer with methanol 20%, to allow transfer small proteins. and also try to make semidry transference.
we run abetta from mammalian cell cultures, and those conditions worked very well.
best,
Thank you very much Dr. Aniko and Dr. Balmaceda for giving me such valuable suggestion. but one thing more i want to know which not directly related to western but protein isolation.
I had tried few buffers for the protein isolation from drosophila head but i am not satisfied. So please suggest me the suitable buffer used for protein isolation from drosophila head for the purpose of western for amyloid beta. please help me by providing complete recipe. thanks in advance.
Hi Sandeep,
You can try immuno-precipitation. I did western blots for amyloid from cell lysates and for detailed procedure look at the following paper.
http://pubs.acs.org/doi/abs/10.1021/mp200530q?prevSearch=omtri&searchHistoryKey=
Good luck
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