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Problem with the SDS-PAGE sample prepaeration?!!


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#1 jalmali

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Posted 16 February 2009 - 03:35 PM

can any body help me please ASAP.....
i tried to prepare my samples for the sds-page, i boiled those samples for 5 minutes at 100C, the samples form an aggregate and a thick gel-like material, which make pippetting the sample is impossible. i don't know if the problem may be due to the boiling step, and if so, what i can do to solve this problem, make the sample solution thin and diluted enough to pippete it in the electrophoresis gel wells.
please tell me any solution for this because i can't throw them and i have to use this sample? i don't know if this write or not, to use 8M urea in SDS buffer to deaggregate the soultuion (i read this in one of the troubleshooting forums)
appreciate your help anyhow...

#2 jiro_killua

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Posted 16 February 2009 - 03:57 PM

can any body help me please ASAP.....
i tried to prepare my samples for the sds-page, i boiled those samples for 5 minutes at 100C, the samples form an aggregate and a thick gel-like material, which make pippetting the sample is impossible. i don't know if the problem may be due to the boiling step, and if so, what i can do to solve this problem, make the sample solution thin and diluted enough to pippete it in the electrophoresis gel wells.
please tell me any solution for this because i can't throw them and i have to use this sample? i don't know if this write or not, to use 8M urea in SDS buffer to deaggregate the soultuion (i read this in one of the troubleshooting forums)
appreciate your help anyhow...


I seem to vaguely remember this happened to me years ago....

but couldn't remember what exactly went wrong

logically, only 3 things could go wrong

1. protein itself
2. lysis buffer used to lyse cells
3. loading buffer

My guess would be loading buffer, maybe the loading buffer is very old and the beta-mecap has evaporated?
(although I haven't ask when you boil your samples, were they already in loading buffer?)

Edited by jiro_killua, 16 February 2009 - 03:58 PM.


#3 jalmali

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Posted 16 February 2009 - 06:39 PM

can any body help me please ASAP.....
i tried to prepare my samples for the sds-page, i boiled those samples for 5 minutes at 100C, the samples form an aggregate and a thick gel-like material, which make pippetting the sample is impossible. i don't know if the problem may be due to the boiling step, and if so, what i can do to solve this problem, make the sample solution thin and diluted enough to pippete it in the electrophoresis gel wells.
please tell me any solution for this because i can't throw them and i have to use this sample? i don't know if this write or not, to use 8M urea in SDS buffer to deaggregate the soultuion (i read this in one of the troubleshooting forums)
appreciate your help anyhow...


I seem to vaguely remember this happened to me years ago....

but couldn't remember what exactly went wrong

logically, only 3 things could go wrong

1. protein itself
2. lysis buffer used to lyse cells
3. loading buffer

My guess would be loading buffer, maybe the loading buffer is very old and the beta-mecap has evaporated?
(although I haven't ask when you boil your samples, were they already in loading buffer?)






thanks mr.enthusiasim for your answer,
but the sample was not with the loading buffer, i read in many internet sites that could happen due to the protein nature(that this protein should not be boiled at 100C degree). anyway, they suggest to add 8M urea with SDS buffer to the sample to deaggregate the formed gel-like material, do you agree? or do you know anything about this?
thanks in advance again....

#4 T C

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Posted 16 February 2009 - 07:50 PM

Hey

Its because of excessive heating...I faced thesame problem once....now I just boil at 80-90 deg C for a minute or so.......SDS in dye is good enough to denature....Kindly check the attachment (ref 18).

TC

Attached Files



#5 jalmali

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Posted 17 February 2009 - 01:55 PM

Hey

Its because of excessive heating...I faced thesame problem once....now I just boil at 80-90 deg C for a minute or so.......SDS in dye is good enough to denature....Kindly check the attachment (ref 18).

TC


thank you man for your help,
i appreciate your help...
take care

#6 mdfenko

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Posted 18 February 2009 - 09:25 AM

you should not boil the sample unless it is mixed with loading buffer. heat will denature the protein and cause it to aggregate. sds and reducing agent will prevent the aggregation as the protein denatures (this, of course, is not the only function of sds and reducing agent in this system).
talent does what it can
genius does what it must
i do what i get paid to do

#7 yobou

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Posted 13 March 2009 - 09:19 PM

I got a similar problem only with nuclear proteins prepared in high salt content buffer upon boiling in sample loading buffer. but by pipetting several times before loading that aggregate dissappeared




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