Protein extraction - Western Blot

Use this category for questions regarding problems manipulating proteins in molecular biology applications (expression, detection, etc.)

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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 16 2016 3:49 pm

you should use anywhere from 0.1% to 0.5% sds.

you can use either the pbs with inhibitors and sds or ripa. ripa is the preferred solubilizer. i recommend that you use it.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 16 2016 4:06 pm

mdfenko wrote:you should use anywhere from 0.1% to 0.5% sds.

you can use either the pbs with inhibitors and sds or ripa. ripa is the preferred solubilizer. i recommend that you use it.


Great. I have a RIPA buffer (borrowed from another lab) but it contains both SDS and Triton. Will this affect in any way to my proteins or the presence of both along with other reagents like Tris, EDTA, EGTA, Nacl, and Na-deoxycholate won't have any bad affect? Should I add halt protease to this too?

Also, after adding the buffer (500-600 µl) and homogenizing the pellet, should I centrifuge it for 20 min at 4˚C at 13,000 rpm like I used to do when I homogenized it with PBS?
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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 17 2016 7:50 am

adding the inhibitors won't hurt.

pelleting should be similar. but, check the pellet after to determine if it was sufficient.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 17 2016 1:18 pm

mdfenko wrote:adding the inhibitors won't hurt.

pelleting should be similar. but, check the pellet after to determine if it was sufficient.


Thanks.
Also, can you please tell me how to decide the gel % for resolving gel depending on the protein size. For example: I have a 121 kda protein and I made a 8% gel. Will this work?
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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 18 2016 7:24 am

8% should be okay but it also depends on the range of protein sizes and the desired resolution.see this graphic:

Image
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Re: Protein extraction - Western Blot

Postby Reuter » Sep 01 2017 2:41 am

That's a helpful graphic, thanks mdfenko.
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