Protein extraction - Western Blot

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Protein extraction - Western Blot

Postby newbie17425 » Nov 15 2016 10:28 am

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.
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Re: Protein extraction - Western Blot

Postby relaxin » Nov 15 2016 5:54 pm

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.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 15 2016 6:26 pm

relaxin wrote: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.


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?
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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 16 2016 12:31 pm

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.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 16 2016 12:51 pm

mdfenko wrote: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.


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?
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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 16 2016 12:57 pm

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).
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 16 2016 1:04 pm

newbie17425 wrote:
mdfenko wrote: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.


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?



Thanks. I don't need to do any more ELISA so Triton is a better option :)
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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 16 2016 1:38 pm

sds would be the better option if you're only using the protein for western blotting.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 16 2016 1:39 pm

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


0.1% SDS? Will adding SDS/Triton and homogenizing the pellet again pull down the concentration by using BCA assay?
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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 16 2016 2:02 pm

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.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 16 2016 2:17 pm

mdfenko wrote: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.


So how do I quantify?
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Re: Protein extraction - Western Blot

Postby mdfenko » Nov 16 2016 4:10 pm

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)
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Re: Protein extraction - Western Blot

Postby relaxin » Nov 16 2016 5:16 pm

newbie17425 wrote: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?


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.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 16 2016 5:19 pm

mdfenko wrote: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)


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.
Last edited by newbie17425 on Nov 16 2016 7:12 pm, edited 2 times in total.
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Re: Protein extraction - Western Blot

Postby newbie17425 » Nov 16 2016 5:32 pm

relaxin wrote:
newbie17425 wrote: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?


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.


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

Thanks.
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