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How to add prepare protein sample for loading into the gel?

sds-page western blot sample buffer loading buffer Bromophenol blue

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#1 nasif

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Posted 05 October 2017 - 12:55 PM

Hey all, before loading the protein into the sds-page well, what should i do to prepare the protein? The protocol my supervisor gave shows, "To 20 µl of sample, add 2 µl of 1 % BPB and 2µl of 1 M DTT. Boil for 5 mins and Load". However, he has been saying about 2X and 6X concentration of the solution. I'm not getting what he meant and at this point I'm too afraid to ask. Can I just add BPB and DTT to the sample and load it?If yes, what should be amount or calculation i should follow?



#2 bob1

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Posted 05 October 2017 - 01:48 PM

The 2x and 6x are concentrations of solutions that contain many ingredients. The 6 indicates parts dilution. For instance with the 2x you would take 1 part lysate and 1 part 2x dye solution to make 1x solution with the lysate. 6x is 5pasrtss lysate to 1 part dye solution. The usual loading dye solution for western blots is known as Laemmli buffer, if that helps you find solutions...

 

A common recipe for 6x solution can be found here.



#3 nasif

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Posted 05 October 2017 - 08:16 PM

The 2x and 6x are concentrations of solutions that contain many ingredients. The 6 indicates parts dilution. For instance with the 2x you would take 1 part lysate and 1 part 2x dye solution to make 1x solution with the lysate. 6x is 5pasrtss lysate to 1 part dye solution. The usual loading dye solution for western blots is known as Laemmli buffer, if that helps you find solutions...

 

A common recipe for 6x solution can be found here.

 

Thanks for the reply. That was helpful. So when to use 2X and when to use 6X for loading? also at what degree Celsius should i boil after mixing? 



#4 mdfenko

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Posted 06 October 2017 - 01:36 PM

you don't mention addition of sds to the sample. if your sample already has sufficient sds in it, then you just need to add reducing agent (dtt) and tracking dye (bpb). you would then "boil" at about 95-100C for up to 5 minutes or at 60-70C for 10-20 minutes.

 

laemmli, or sample, buffer contains buffer, sds, reducing agent (dtt or 2-me) and tracking dye.


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