4 replies to this topic

[Always&Forever]

Hi all,
i am new in the forum but from what i have seen so far, i hope that may be you could help me.

So this is my problem:
I am dealing with retina tissue and i am looking for a phosphorylated protein.
I do western blot analysis.
So far i was using sonication to homogenize my protein but from discussions that i had with some fellows in the lab, they told me that may be sonication is not a good way to "treat" my retinas and therefor my protein.

Does anyone have worked with retina tissue?
Do you know which is the best way to homogenize retinal tissue?
If sonication is not a problem, which settings do you recommend?

*The only reason i am not grinding my tissue is that retina is really tiny and i will lose a huge amount of it on the grinder.

Thank you in advance and looking forward for your feedback,

Always&Forever

[bob1]

Sonication will disrupt the protein, making it difficult to detect on the blot.  I would try snap-freezing your tissue in RIPA buffer with protease inhibitors and then thawing, repeat a couple of times and then blot from there.

[Aris]

bob1, on May 26 2009, 04:40 PM, said:

Sonication will disrupt the protein, making it difficult to detect on the blot.  I would try snap-freezing your tissue in RIPA buffer with protease inhibitors and then thawing, repeat a couple of times and then blot from there.

Hey Bob1, got a follow up question on that. Sonication is widely used in almost all labs around the world. If as you say thsi will disrupt the protein then why do we use it? Besides that, the incerasing freeze thaw steps will also cause the protein to degenerate. Am i right or wrong?

[Always&Forever]

bob1, on May 26 2009, 05:40 PM, said:

Sonication will disrupt the protein, making it difficult to detect on the blot.  I would try snap-freezing your tissue in RIPA buffer with protease inhibitors and then thawing, repeat a couple of times and then blot from there.

Hi bob1. Thanks for your reply but i have a problem.
I have already put my retinal tissue in lysis buffer which contains Tris-HCl, NaCl, CaCl2, NaN3, TritonX -100, NaPPIs, B-glycerophosphate, Na3VO4,
NaF ,DTT and Protease inhibitors.  Can i try the snap-freezing and thawing procedure?

I have tried the snap-freezing and thawing procedure before with different tissues but it didn't work well:
i compared with protein assay sonicated tissue and tissue that was freezed and thawed and i had more protein at my sonicated samples.  
Of course i didn't use the RIPA buffer that you are mentioning.


Thank you so much for your time and help,
Always&Forever

[bob1]

Aris, on May 26 2009, 07:27 PM, said:

bob1, on May 26 2009, 04:40 PM, said:

Sonication will disrupt the protein, making it difficult to detect on the blot.  I would try snap-freezing your tissue in RIPA buffer with protease inhibitors and then thawing, repeat a couple of times and then blot from there.

Hey Bob1, got a follow up question on that. Sonication is widely used in almost all labs around the world. If as you say thsi will disrupt the protein then why do we use it? Besides that, the incerasing freeze thaw steps will also cause the protein to degenerate. Am i right or wrong?

Freeze/thaw will also degrade the protein, but in my experience (limited with sonication, I will admit) less degradation than by sonication.  I think the main problem with sonication is the formation of bubbles that lead to rapid oxidation of the protein.

Always&Forever, on May 27 2009, 07:49 AM, said:

I have already put my retinal tissue in lysis buffer which contains Tris-HCl, NaCl, CaCl2, NaN3, TritonX -100, NaPPIs, B-glycerophosphate, Na3VO4,
NaF ,DTT and Protease inhibitors.  Can i try the snap-freezing and thawing procedure?

I have tried the snap-freezing and thawing procedure before with different tissues but it didn't work well:
i compared with protein assay sonicated tissue and tissue that was freezed and thawed and i had more protein at my sonicated samples.  
Of course i didn't use the RIPA buffer that you are mentioning.

It should be fine in your buffer, RIPA is just a standard western lysis buffer.  It looks like you have a fair range of protease and phosphatase inhibitors in there, so it should work.  If you get better results with the sonication, stick with that.