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How do minimize influence of TissueTek(OCT) in Ripabuffer samples for SDS-Page


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#1 winnyee

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Posted 09 August 2012 - 09:42 AM

Dear Forumusers,

It seems that we could have a problem. After cryosectioning of samples embedded in tissue tek, protein extraction of residual tissue and sections were made in ripa-buffer and protein measurement was done by BCA. Samples were loaded and SDS-Page was carried out. Signals were found for proteins lower than about 80-100 kDa, over 100 kDa no signals were found and a kind of shadow was visible on the recorded images in this blot area. It seems that especially the samples with more tissue-tek left lead to this effect.

Had anyone similar problems and maybe also solved these ?

Thanks a lot for your help !!!

#2 mdfenko

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Posted 09 August 2012 - 12:25 PM

more information is required

what is acrylamide concentration in sds-page?

transfer buffer composition?

etc.
talent does what it can
genius does what it must
i do what i get paid to do

#3 winnyee

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Posted 10 August 2012 - 04:38 AM

These effects are found with gels wit 7.5 as well as 12 % acrylamide. The transfer buffer composition for wet blotting in a tank was:

6,06 g Tris = 25 mM
28,83 g Glycin = 192 mM
400 mL Methanol
7,5 mL 10% SDS =
residual H2O added to total volume of 2 L

In general it seems that the tissue-tek amount inhibits antibody binding to the targets over 100 kDa. The problem is maybe to get rid off the tissue tek within samples ? If this is the case, how can be this achieved or is it really a blotting problem ? Interestingly, no residual bands on the gel were found after blotting and Coomassie staining (only few, weak blue bands at maximum molecular weight over 200 kDa).
What we also see that in samples with hardly tissue tek the signals for the targets over 100kDa are well detectable.

Do you need further information ?

best wishes

#4 mdfenko

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Posted 11 August 2012 - 09:47 PM

have you tried staining the membrane with ponceau s? staining the gel, after transfer, with silver?
this can help you determine where your protein is.

i don't know anything about tissue tek, but if your proteins show up with less present then maybe you can reduce the concentration in your samples prior to addition of loading buffer (by drop dialysis or some other method).

by the way, you can add up to 10ml 10%sds to 2000ml of transfer buffer (0.05% final).
talent does what it can
genius does what it must
i do what i get paid to do




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