6X Laemmli buffer

This post was written by admin on March 5, 2009
Posted Under: Buffers & recipes,Western blot

Laemmli is a sample buffer to use in western blot.  6X sample buffer is added to each protein sample and is boiled or heated for 5-10 minutes.

Recipe to prepare 10 ml:

- 1.2gr SDS (sodium dodecyl sulfate)
- 6mg bromophenol blue
- 4.7ml glycerol
- 1.2ml Tris 0.5M pH6.8
- 2.1ml dwater
warm it a little bit and shake it till everything is dissolved. It takes time.
- add 0.93gr DTT.
Wait until it is completely dissolved.

Aliquote and keep frozen at-20ºC.

Reader Comments

Hi, can you please clarify the dilution ratio of 6xLaemmli buffer with protein sample? My understanding is 5:1 = Protein sample:buffer. Is that right?

thanks

#1 
Written By tengku on August 24th, 2009 @ 4:46 am

Hi, Yes, you are totally right. 5ul of protein sample + 1ul of 6X Laemmli buffer. Hope it works!

#2 
Written By admin on August 24th, 2009 @ 10:27 am

Hi! I was trying to solve SDS, but it seems, that it don’t want to be solved :) Please, any suggestions. Thanks in advance

#3 
Written By Lev on September 17th, 2009 @ 5:59 am

Hi, you want to make 10ml 6X Laemmli buffer, but From your receipe, the total volume just 8ml. What should add more? Just whater? Thanks!

#4 
Written By hui chen on September 17th, 2009 @ 9:32 am

Hi Hui Chen, thanks for your comment. When you add the SDS, bromophenol and DTT, the volume increases to about 10ml. You won’t need to add more water.

#5 
Written By admin on September 17th, 2009 @ 11:22 am

Hello Lev, It takes a little bit to dissolve. What I normally do is to weight the SDS and put it together with the water in a flask, throw a magnet in it and leave it shaking on a stirring hot plate. Meanwhile I weight the rest of the things in the recipe and add them to the flask progressively. The plate doesn’t need to be very hot, just a litlle bit warm to help SDS dissolve. Don’t let it boil. I hope it works!

#6 
Written By admin on September 17th, 2009 @ 11:26 am

Hi!Is possible to use beta mercaptoethanol instead of DTT, if so, how many ml I must add to your 6X sample buffer? thanks!

#7 
Written By Laura on October 28th, 2009 @ 6:34 am

I want to keep the total protein amount I load in all my western blot wells the same; the different samples I am testing have differing protein concentrations therefore I will require different final sample volumes. Which means, I think, that I will also have different amounts of Laemmli buffer in each well. Is this correct? And does it matter if there are different volumes in each well as long as the maximum ammount is not exceeded? And does it matter if there are different amounts of Laemmli buffer?
I’ve also noted some labs use 2X Laemmli buffer – why?
Thank you
Tatiana

#8 
Written By Tatiana Christides on October 30th, 2009 @ 1:30 pm

hello sir i want to know that what is the ratio of sample buffer and protien sample…….when the sample buffer concentration is 6x?????

#9 
Written By deepak tripathi on November 13th, 2009 @ 10:51 pm

Do you mind listing out the final concentrations of the reagents (such as: 12% SDS, 0.06& BB, etc…)?

Thanks for your post!

#10 
Written By Justin on December 20th, 2009 @ 10:24 am

Hi I want to use the 6* Laemli for SDS-PAGE with native proteins. What is the DTT doing here. I thought it was an inhibitor ?? Can I leave it out and replace volume with UP-water ??
Thanks

#11 
Written By Betty on March 23rd, 2010 @ 12:48 am

Could some body explain to me what is the difference using 2X Laemmli buffer vs 5X Laemmli buffer ?Many thanks

#12 
Written By Viki on May 1st, 2010 @ 10:09 am

hi
keeping SDS to sit and dissolve on a magnetic stirrer will generate lots of bubbles ! that is a real non-sense when adjusting the volume. I will prefer to add SDS that is already in a solution( as 20% or 10% ready to go from Gibco or SIGMA). That will both save time and rescue from un-wanted bubbles.
Also better to add DTT/or Beta-mercaptoethanol “fresh” to the Lamelli. BME degrades at room temp as well as slowly in -20 when in solution. Better to add fresh right before you use.

#13 
Written By subhradip on May 8th, 2010 @ 2:25 pm

thank you, is this suitable for liquid protein sample?

#14 
Written By mallikarjun on May 31st, 2010 @ 6:21 am

Hi, I have a question regarding the 6x Laemmli buffer. After you make it, aliquote and freeze, how many times you can thaw and freeze the buffer? Does the DTT goes bad?

#15 
Written By Mariana Franco on August 9th, 2010 @ 6:13 am

i have a purchased laemmli buffer, but i didnt knw it had to be stored at -20C. i had refrigerated it,and now it has some flakes in it….can it still be used????

#16 
Written By Asna on August 12th, 2010 @ 9:09 am

hi…i am using a ready made laemmli buffer,,,i didnt know that we had to store it at -20C….it was stored at 4C and now it has some flakes in it….can it still b used??????? =(

#17 
Written By asna on August 14th, 2010 @ 1:25 am

Hi, I want know the how much 6x or 2x lamelli buffer will be needed to denature 1 mg of protein…………

#18 
Written By ajit on August 20th, 2010 @ 8:43 pm

Here you will find a protocol for 4X Seperation buffer for SDS-PAGE: http://www.elabprotocols.com/viewer/?id=131

#19 
Written By eLabProtocols on September 14th, 2010 @ 11:29 pm

hello everyone

I would like to ask you wich is the range of DTT for denaturing condition. There are several concentration on internet.

#20 
Written By Nelson on October 26th, 2010 @ 4:13 pm

Hi
Can Laemmli sample buffer(2X) be used to lyse the cells in culture?If so, the bromophenol blue be added later after the OD is measured for protein concentration

#21 
Written By Azfer on February 16th, 2011 @ 7:15 am

Why is there no 2-mercaptoethanol?

#22 
Written By Susan on March 14th, 2011 @ 11:11 am

Hi, if u wanna substitute dtt for b-mercaptoethanol, is it still the same amount you listed?

Thanks!

#23 
Written By Pete on May 9th, 2011 @ 8:03 am

Hy Everybody

I would like to know, if i can use this buffer leamly 6X to do a total protein extraction from Nicotiana Bentamiana,

tks

#24 
Written By Naou on May 20th, 2011 @ 2:24 am

thanks

#25 
Written By Helen on August 6th, 2011 @ 8:48 am

Hi,Happy friendship day i want a total protein isolation protocol for wheat to carry out protein fingerprinting of various variety of wheat

#26 
Written By Anant on August 7th, 2011 @ 1:37 am

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#27 
Written By jason on December 16th, 2011 @ 6:34 pm

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#28 
Written By Theressa Schellin on December 17th, 2011 @ 8:41 am

I need to make a 5X of SDS buffer. I currently have a protocol for 2x and if I add equal volume of dh20 to that I get a 1x. How do I calculate what I need to make the 5x solution.

Can I multiply my 2x protocol by 3 to get a 5x stock solution?

Your help would be greatly appreciated

#29 
Written By Melody on January 26th, 2012 @ 1:58 pm

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