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-   -   SDS Boiling of Samples Any Alternative Methods? (http://www.molecularstation.com/forum/sds-page-gel-electrophoresis-forum/3568-sds-boiling-samples-any-alternative-methods.html)

joey20 07-11-2008 10:46 PM

SDS Boiling of Samples Any Alternative Methods?
 
i hate boiling my samples as i get aggregates of proteins running near the top of the gels. also if you are using specific dyes/probes etc you want hooked to the protein, boiling kills/knocks off these guys.....is there any way to not boil your samples before sds page?

danfive 07-14-2008 07:18 PM

Re: SDS Boiling of Samples Any Alternative Methods?
 
How about native PAGE (non-reducing conditions)?

aftabac 07-17-2008 05:41 AM

Re: SDS Boiling of Samples Any Alternative Methods?
 
So Far i could not find any alternative method of boiling in sample preparation for SDS-PAGE. if somebody know then he should share with us, so we can make a compartive analysis of both procedures.


regards
aftab

keithbrent2000 08-05-2008 04:28 PM

Re: SDS Boiling of Samples Any Alternative Methods?
 
You could try just not boiling at all to see how the boiled sample compares with the unboiled sample. You could also heat your sample at 37 degrees Celsius for longer (maybe 10 minutes), so that your sample isn't actually boiling. Another possibility could be to include some urea (maybe like 2M) in the sample.

saprathap 11-14-2008 04:33 AM

Re: SDS Boiling of Samples Any Alternative Methods?
 
Quote:

Originally Posted by joey20 (Post 8386)
i hate boiling my samples as i get aggregates of proteins running near the top of the gels. also if you are using specific dyes/probes etc you want hooked to the protein, boiling kills/knocks off these guys.....is there any way to not boil your samples before sds page?

:thumbs:

admin 11-16-2008 04:09 AM

Re: SDS Boiling of Samples Any Alternative Methods?
 
I have read somewhere boiling at 100 C is bad (most people set it at 100 on the heating block and 5 minutes!), and 91 C - 93 C is improved.

Sorry I forgot which paper I read it but try it during your optimization. A few samples at 100 C and some at 90 - 93 C. Let us know what you get, I would actually use 91 C for 3 minutes.

cheers

butters 12-09-2008 02:51 AM

Re: SDS Boiling of Samples Any Alternative Methods?
 
the aggregates appear even after centrifuge/spin down? i forgot to mention that u only take the supernatant right? maybe you can show us the gel and how does the aggregates look like.

lja 12-12-2008 03:48 PM

Re: SDS Boiling of Samples Any Alternative Methods?
 
"Membrane protein crystallization and purification", eds. C. Hunte, G. von Jagow, and H. Schaegger, pag.93 they suggest 40C for 30', which I use also for the soluble fraction of my cells. Urea variates the molecular weights, at least to my knowledge. I hope it is useful!
cheers

JerryWang 02-05-2010 01:22 AM

Re: SDS Boiling of Samples Any Alternative Methods?
 
I do 96 degrees for 8 mins on the PCR thermocycler for my 96 wells =) works fine when i run 6 gels at a time

tgd 02-08-2010 03:20 PM

Re: SDS Boiling of Samples Any Alternative Methods?
 
Hello,

As far as I know, boiling was introduced to combat proteolysis. The idea being that as SDS unfolds the proteins in the sample any protease there will have a 'field day' for the time spent at near optimum temperature with an unfolded substrate to act on. It is essential, therefore, to get the sample to a temperature where any proteases present are instantly inactivated. Probably, there is no need to go to 100 degrees C, but 40 degrees C for 30 min seems crazy to me, unless the risk of proteolysis can be completely excluded.

For this reason also, you should always add HOT sample buffer to the sample, especially with crude extracts.

There are a few other points. Do you centrifuge you samples prior to loading? This usually gets rid of precipitation.

Do you have a stacker gel? It is not uncommon for high molecular weight proteins not to enter the gel. But if you have a stacker (say 4 percent or something like that) this is an unlikely explanation.

Is there a lot of nucleic acid present? Are your samples 'gloopy'. If this is the case, you may need to sonicate.

I suppose the answer is there is no need to boil, but beware of proteolysis

Good luck,
tgd


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