Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Basic Lab Protocols and Techniques > SDS-PAGE Gel Electrophoresis Forum
Register Search Today's Posts Mark Forums Read

SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


SDS Boiling of Samples Any Alternative Methods?

SDS Boiling of Samples Any Alternative Methods? - SDS-PAGE Gel Electrophoresis Forum

SDS Boiling of Samples Any Alternative Methods? - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 07-11-2008, 10:46 PM
Pipette Filler
Points: 2,407, Level: 31 Points: 2,407, Level: 31 Points: 2,407, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2006
Posts: 13
Thanks: 0
Thanked 0 Times in 0 Posts
Default 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?
Reply With Quote
  #2  
Old 07-14-2008, 07:18 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: SDS Boiling of Samples Any Alternative Methods?

How about native PAGE (non-reducing conditions)?
Reply With Quote
  #3  
Old 07-17-2008, 05:41 AM
aftabac's Avatar
Nobel Laureate
 
Join Date: Jun 2006
Location: Pakistan
Posts: 570
Thanks: 5
Thanked 41 Times in 34 Posts
Default 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
Reply With Quote
  #4  
Old 08-05-2008, 04:28 PM
Pipette Filler
Points: 731, Level: 14 Points: 731, Level: 14 Points: 731, Level: 14
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2008
Posts: 14
Thanks: 0
Thanked 0 Times in 0 Posts
Default 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.
Reply With Quote
  #5  
Old 11-14-2008, 04:33 AM
Pipette Filler
Points: 196, Level: 3 Points: 196, Level: 3 Points: 196, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Location: Tamilnadu,India
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: SDS Boiling of Samples Any Alternative Methods?

Quote:
Originally Posted by joey20 View Post
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?
Reply With Quote
  #6  
Old 11-16-2008, 04:09 AM
admin's Avatar
Administrator
 
Join Date: Nov 2005
Posts: 1,418
Thanks: 883
Thanked 68 Times in 58 Posts
Default 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
Reply With Quote
  #7  
Old 12-09-2008, 02:51 AM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default 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.
Reply With Quote
  #8  
Old 12-12-2008, 03:48 PM
lja's Avatar
lja lja is offline
Pipette Filler
Points: 163, Level: 3 Points: 163, Level: 3 Points: 163, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Posts: 4
Thanks: 1
Thanked 1 Time in 1 Post
Default 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
Reply With Quote
The Following User Says Thank You to lja For This Useful Post:
kdwyer (05-01-2009)
  #9  
Old 02-05-2010, 01:22 AM
Pipette Filler
Points: 32, Level: 1 Points: 32, Level: 1 Points: 32, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2010
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default 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
Reply With Quote
  #10  
Old 02-08-2010, 03:20 PM
tgd tgd is offline
Volunteer
Points: 1,303, Level: 21 Points: 1,303, Level: 21 Points: 1,303, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Posts: 45
Thanks: 2
Thanked 22 Times in 14 Posts
Default 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
Reply With Quote
The Following 2 Users Say Thank You to tgd For This Useful Post:
admin (05-15-2010), danfive (04-21-2010)
Reply

Tags
alternative , boiling , methods , samples , sds


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
molecular biology laboratory from scratch RE: Methods Digest, Vol 40,Issue 17 Utsugi, Heidi K Protocols and Methods Forum 0 09-23-2008 05:50 PM
Resource: Mass Spectrometry-based Methods of Proteome Analysis danfive Protein Science 0 06-02-2008 08:03 PM
Methods Digest, Vol 34, Issue 23 Protocols and Methods Forum 1 03-27-2008 10:57 AM
Methods Digest, Vol 17, Issue 23 Sayyari, Mohammad Protocols and Methods Forum 0 10-25-2006 09:11 AM
Methods Digest, Vol 3, Issue 4 TieQiao Wu Protocols and Methods Forum 0 08-05-2005 01:03 AM


All times are GMT. The time now is 12:12 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.18514 seconds with 16 queries