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Sample Buffer Recipe (Laemmili)


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#1 Mummytomax

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Posted 01 March 2005 - 09:02 PM

Hi,
I'm trying to find the rhyme and reason behind the sample buffer recipe's we use? All are designed to do the same thing but all have different proportions of the same ingrediants. After looking on the web this afternoon I figure I may as well chuck them all in in any amount and it'll work (but we all know it doesn't happen B) )

The 2x recipe I used for 3 years during my PhD in the 90's was 0.5M Tris-HCl pH 6.8, 5% glycerol, 2% SDS and 100 mM DTT without a problem.
Now my antibody company suggested for 2x buffer was 125 mM Tris-HCl, 10% glycerol, 10% SDS, 130 mM DTT.
A friend uses 3 x buffer with 180 mM tris-HCl, 20% glycerol, 6% SDS, 125 mM DT.

Two protocols I looked at on the protocol website stated different amounts again with one using tris at pH 8 instead.

Which is one to beleive and why do we all use different recipes (i.e why is there not a standard one?) Is glycerol there for something other than weighing the sample down into the well? Does increasing the SDS increse the seperation of the protein? Is this the same for DTT????

Ideas?

This is aimed to be more of a discussion topic - I'm not looking for an answer! I'm happy with the recipe that works for me :P

#2 fred_33

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Posted 02 March 2005 - 02:08 AM

hi

i think that, as long as your recipe is functionning, there's no need to change it B)

#3 Simonsays

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Posted 02 March 2005 - 06:01 AM

The glycerol is only there so the prots go down the well within the hour!!
SDS binds to the proteins (1.3g of SDS/ g of prot) and give them the negative charge necessary to be able to make a linear (or logarithmic) relation between mass and migration. The DTT is there to reduce any S-S bond that could provide a secondary/tertiary structure and/or dimer formation.
My recipe isn't like anyone your described!! Fred_33 and are right... as long as it works!!

Simon

#4 badcell

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Posted 02 March 2005 - 07:31 AM

The standard of the Laemmli sample buffer is in Laemmli original paper (Nature 227:680-5, 1970). But as Simon and Fred said, any recipe will do as long as it works. Essentially, it has to have glycerol to give weight to the sample, SDS to give it negative charge, and DTT or b-mercaptOH in case you want reducing conditions.
As for the buffer, it should be Tris (any concentration on the mM range will probably work)- but the important thing it that it should be pH 6.8. I don't believe it would work with Tris pH 8, as you mention you saw in some recipe. For the stacking gel to stack the proteins, the pH of the gel and the sample should be the same. That's what I was taught, at least; I've never tried it.
Cheers!
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)

#5 Mummytomax

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Posted 02 March 2005 - 08:29 PM

You were taught well!

7 years of western blotting and I just follwed the recipe I was given without asking 'how this sample buffer works' before..(oh I knew the DTT bit). ^_^




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