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Sample prep. for 2D-PAGE service - (Sep/06/2006 )

Hi, does anyone know if i could use 2XLB (loading buffer) treated cell lysates directly for 2D-GEL runing?
By the way, are there any company that provides 2D-GEL services in Maryland?

Thanks in advance! rolleyes.gif

-zhangt1-

no. you cannot run ief (first dimension) with sds treated protein.

-mdfenko-

QUOTE (mdfenko @ Sep 7 2006, 07:46 AM)
no. you cannot run ief (first dimension) with sds treated protein.


Thanks a lot for your info!

the other problem is can i run SDS-PAGE (one dimension) gel using protein extractions containing urea with common laemmli sample buffer? just before 2D-gel, i want to check with immunoblot if lysis buffer specific for IEF can still extract my target protein.

in other words, can samples prepared for IEF be used directly on SDS-PAGE, if so, are there any other treatment needed?

Thanks again and i am dying for your reply!

-zhangt1-

how do you perform? the samples are solved in IEF rehydration buffer containing urea and some percent of detergents such as CHAPS?; will you add multiple concentrated Laemmli-buffer to your probes? urea should be no problem for SDS-PAGE, may be other detergents than SDS are more critical as micelles may disturb SDS effect

-The Bearer-

as kosmodrom says, urea is no problem for your sample. the only thing you have to be careful about is added detergents. triton x-100, nonidet p-40 and other, similar, non-ionic detergents tend to displace sds from the protein and will, therefore, interfere with the migration of the sample.

-mdfenko-

QUOTE (kosmodrom @ Sep 7 2006, 01:44 PM)
how do you perform? the samples are solved in IEF rehydration buffer containing urea and some percent of detergents such as CHAPS?; will you add multiple concentrated Laemmli-buffer to your probes? urea should be no problem for SDS-PAGE, may be other detergents than SDS are more critical as micelles may disturb SDS effect


I mean i'll use the lysis buffer specific for IEF to treat cells, after centrigugation, i collect supernatant and add 2x or 5x laemmli buffer into it, then after 5min, 100 centrigrade denaturation, run the sample on SDS-PAGE and immunobloted with antibody.
Is that practicable?

-zhangt1-

QUOTE (mdfenko @ Sep 7 2006, 04:05 PM)
as kosmodrom says, urea is no problem for your sample. the only thing you have to be careful about is added detergents. triton x-100, nonidet p-40 and other, similar, non-ionic detergents tend to displace sds from the protein and will, therefore, interfere with the migration of the sample.


But it's said that to avoid modification of protiens, never heat a sample after adding urea. when the sample contain urea, it must not be heated over 37 C, elevated termperatures cause urea to hydrolyze to isocyanate, which modifies proteins by carbamylation. So, should i heat samples for SDS-PAGE, or just add laemmli buffer and run SDS-PAGE directly?

-zhangt1-

QUOTE (zhangt1 @ Sep 8 2006, 03:44 PM)
But it's said that to avoid modification of protiens, never heat a sample after adding urea. when the sample contain urea, it must not be heated over 37 C, elevated termperatures cause urea to hydrolyze to isocyanate, which modifies proteins by carbamylation. So, should i heat samples for SDS-PAGE, or just add laemmli buffer and run SDS-PAGE directly?

i often just add sample buffer and run without boiling. most of the time, especially with fresh preparations, there is no difference in apparent mobility but you will have to determine for your own protein.

also, carbamylation may make a significant difference in ief but it shouldn't in sds-page.

-mdfenko-