Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Loading Buffer, Laemli stain

Loading Buffer, Laemli stain - Protocols and Methods Forum

Loading Buffer, Laemli stain - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-19-2005, 03:08 PM
Ippokratis Messaritakis
Guest
 
Posts: n/a
Default Loading Buffer, Laemli stain



Hello all,
I have tried many recipes that can be found through the net on how to
make a Laemli buffer for transferring proteins onto nitrocellulose but
most of them failed to give me the wanted result.
Do u have any valid recipe of making it?

Thanx

Ippokratis Messaritakis
Microbiologist, PhD Student
University of Crete, Faculty of Medicine
Dept of Bacteriology, Parasitology, Zoonoses and Geographical Medicine
Vasilika Vouton
71100 Heraklion, Crete
Greece
Tel. +30 2810 394572
Fax. +30 2810 394740


Reply With Quote
  #2  
Old 10-20-2005, 04:25 PM
Jose de las Heras
Guest
 
Posts: n/a
Default Loading Buffer, Laemli stain


"Ippokratis Messaritakis" <[Only registered users see links. ].uoc.gr> wrote in message
news:[Only registered users see links. ].n et...

what problem are you finding?

Jose


Reply With Quote
  #3  
Old 10-25-2005, 01:12 PM
Dr Engelbert Buxbaum
Guest
 
Posts: n/a
Default Loading Buffer, Laemli stain

Ippokratis Messaritakis wrote:


Does your problem occur during loading, staining or blotting? You seem
to be mixing up a couple of things here.

There are prestained molecular weight markers available, which allow you
to monitor correct separation during electrophoresis.

Once that works satisfactory, you have to stain your gel to verify that
you have bands in your sample. Since you want to do a Western afterwards
you need a non-denaturing stain, (copper, zink-imidazole or - slightly
more sensitive but much more expensive - one of the Sypro fluorescent
stains offered by Molecular probes).

If that worked without misshap, you can blot your gel, there are
essentially two procedures for this: that described by Towbin et al.
(Laemli running buffer without SDS, methanol may be added for better
transfer of small proteins) and that described by Dunn (NaHCO3/Na2CO3).
Of those the latter is cheaper, usually gives better results and is less
well known. Transfer can be either in a tank system or semi-dry, I get
much better results with the tank.

Proper transfer is verified by staining the membrane with Ponceau red.
The gel may be stained with Coomassie, if there are bands left, transfer
time needs to be increased. If the gel is clear but no bands are on the
membrane, you have most likely assembled the sandwich the wrong way
round, with the gel, rather than the membrane facing the positive
electrode.

If that hurdle has been taken too you can do the immunostaining for
specific proteins, at least in your positive control (you did include
both positive and negative controls?) you should see the band in
question.

If you go about this in a logical, step-by-step manner, your instructor
and we will be able to offer specific advice in case of problems.
Reply With Quote
Reply

Tags
buffer , laemli , loading , stain


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
protein purification magnificientleo Proteomics Forum 11 12-29-2010 09:01 AM
SDS-PAGE Protocol moleculardude Protein Science 14 02-12-2010 11:27 AM
Production of Monoclonal Antibodies - Technique in Mouse Cellbiogal Antibody Forum 2 01-08-2009 10:52 PM
Cresol red for PCR loading buffer... Tim Fitzwater Protocols and Methods Forum 1 08-05-2004 05:47 PM
Cresol red for PCR loading buffer... jo Protocols and Methods Forum 1 08-04-2004 10:25 PM


All times are GMT. The time now is 08:46 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13243 seconds with 16 queries