Protein extraction - Western Blot

Go to the profile of Chinmaya Sadangi
Nov 15, 2016
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15

Hi,

I have a doubt regarding protein extraction for Western Blot. I'm using the same protein samples that I used for ELISA and for ELISA I used 1 ml of PBS and 10 µl of halt protease inhibitor to grind the brain followed by high speed centrifugation at 4˚C. I used this method for ELISA and it worked.

As I had a lot of protein extract left, I used it for Western Blots. I tried doing it with TLR9 antibody and I saw the specific bands. I also ran a Coomassie staining to see if there is a protein degradation but found that proteins were good.

My question is: Will there be any problem when I publish as I haven't used the RIPA buffer to lyse the hippocampi and instead used a PBS + Halt protease inhibitor combination?

Thanks.

15 Comments

Go to the profile of relaxin
relaxin 5 months ago

TLR9 is a transmembrane protein. So your buffer will not extract it. So it is difficult to prove that the protein band you detected on Western is actually TLR9. The same question will be asked for your ELISA results. Some membrane proteins do have their ectodomains released from cell surface as soluble proteins (ectodomain shedding). I am not sure if TLR9 belongs to this class of proteins. It boils down to the specificity of your antibody.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

Thanks. I did ELISA for cytokines, not for TLR9. What can I do to for the lysis? I cannot take more animals for WB. Is there any way I can improve the brain homogenate I have for WB?

Go to the profile of relaxin
relaxin 5 months ago

The supernatant of your brain homogenate contains only soluble cytosolic proteins. There is not much you can do. If you have leftover tissues or have saved the pellets of the homogenates, you can re-extract with RIPA buffer and use the extract for Western blot.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

Yes, I have the pellets from the homogenates. I will try re-extracting them and doing WB again.

Thanks.

Go to the profile of mdfenko
mdfenko 5 months ago

you can include some (~0.1%) non-ionic surfactant (eg triton x-100) to the extraction medium to disrupt the membrane. you can also add sds if you don't mind denaturing your protein of interest.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

Thank you for the nice suggestions.

Should I add (Triton/SDS) it to the extracted protein, centrifuge it again, take the supernatant in a new tube, and continue with Coomassie/WB?

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

Thanks. I don't need to do any more ELISA so Triton is a better option

Go to the profile of mdfenko
mdfenko 5 months ago

no. extract from the insoluble pellet.

by the way, triton will counteract the sds. i had meant to say that you could use it instead of triton if you didn't mind denaturing the protein (not a problem for western, problem for elisa).

Go to the profile of mdfenko
mdfenko 5 months ago

sds would be the better option if you're only using the protein for western blotting.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

0.1% SDS? Will adding SDS/Triton and homogenizing the pellet again pull down the concentration by using BCA assay?

Go to the profile of mdfenko
mdfenko 5 months ago

sds instead of triton, not with triton.

sds may have an effect on bca (so may triton).

homogenizing the pellet again, in the presence of a detergent, will release additional proteins into solution.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

So how do I quantify?

Go to the profile of mdfenko
mdfenko 5 months ago

some of the ways:

remove the detergent from enough to perform your protein assay by dialysis or detergent binding spin column

use one of the detergent specific protein assays (bio-rad has a bradford and, i think, a bca that are made for use with detergents and reducing agents

pierce has a fluorescent protein assay that is unaffected by detergent and reducing agents (https://www.thermofisher.com/order/catalog/product/R33200?ICID=search-product)

there is an IR spectrometer that is made for these types of determinations (http://www.emdmillipore.com/US/en/life-science-research/protein-detection-quantification/direct-detect-spectrometer/NWGb.qB.NtgAAAFBfBwRRkwm,nav)

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

I found that Thermo Fisher Scientific BCA assay kit is compatible with SDS.
What % of SDS should I use to resuspend my samples?
Also, should I add PBS and halt protease inhibitor again with SDS? Or can I use RIPA buffer containing SDS and Triton with halt protease?
Kindly advice.

Go to the profile of mdfenko
mdfenko about 1 month ago

sorry that i didn't see your last question until now, i hope this isn't too late...

the protein assay kit tells you the most detergent that can be used. however, i would use 0.1% sds. i wouldn't use sds and triton together, triton will displace sds from the protein.