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5x SDS lysis buffer

lysis buffer protein

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5 replies to this topic

#1 JoeSus

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Posted 31 May 2013 - 09:44 AM

Hi,

I am trying to substitute 1% triton for 1% SDS in my lysis buffer, but while the 5x concentration of Triton-X 100 was not viscious at all, the 5% SDS solution is extremely viscious and hard to work with. I just wanted to check this was normal and that there wasnt any weird interactions going on, or it is not normal and I have made an error somewhere. The concents of 100ml lysis buffer were:

12.5ml Tris-Hcl pH 7.4
33.33 Sodium Fluroide
10ml NaCl
10ml EGTA
1ml EDTA
25ml Na pyrophosphate
9ml water

5g SDS

Thanks alot for any help
Joe

#2 Curtis

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Posted 31 May 2013 - 07:07 PM

5 g in 10 ml? that's like more than half of the volume, right? You could have used NP40 instead.

#3 JoeSus

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Posted 01 June 2013 - 03:12 AM

Hi Curtis, thank you for your reply. Sorry I should have been more clear, thats the entire recipe so everything in total adds to just under 100ml before adding the 5g SDS. I've thought about using NP40 but I am using the protein product for gel electrophoresis and didnt want to have SDS competing with NP40/ we were told by the company providng our antibodies for western blot to solubilise in SDS. Thanks again for your reply.

#4 Curtis

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Posted 01 June 2013 - 07:35 AM

Sorry i thought total volume is 10 ml and I got confused. Are your solutions cold when you make the buffer?

#5 JoeSus

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Posted 01 June 2013 - 09:07 AM

Yea I was worried about that so I mixed them with the magnetic spinner on a hotplate set to low heat to try and get it into solution, but it was still very viscous. I have read conflicting things as to whether precipitated SDS forms a viscous solution, but I guess it depends on what else is present in the mix. It is a 5X buffer so hopefully after dilution it will become less viscous, otherwise Ill look for a new SDS lysis buffer! Thanks again for your response.

#6 Curtis

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Posted 01 June 2013 - 11:09 AM

The sds-page loading buffer in western blot is usually 6x but it's not viscous. However there is a solution called solution 2 that we use for plasmid extraction , and this solution got sds and NaOH inside only. I've noticed whenever i make this solution and put it on ice or a cold place it becomes so viscous that i have to make another one. I do 't know the product of this combination, but as you mentioned, definitely the other chemicals in the mix are also important.





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