Western Protocol Details

[cyp_sci]

Hi everyone,
I just started doing western blotting and I feel so confused as to what is for what. I mean why do we choose PBS/t over TBS/t over a given instance? What are the use of the ingredients? Why do we put glycerol in the loading buffer? I will welcome any technical/biochemical insights you will have in the whole western method!

Thanks a lot

[admin]

Hello Cyp_sci,
that is an interesting question.

I guess its optimization and trial n error. Most people use TBST, however some found that PBS gave better results for a given protein/antibody.

Also glycerol is used to make the sample "heavy" and more dense. This is to keep it from being lost when loaded into the gel.

Any other questions you have may be answered by others or me We have done too many westerns lol although its impossible to know everything so im still learning.

cheers

[cyp_sci]

Hello,
Thanks for the prompt answer.
As I said I am new to the western world, and I have problems with loading the sds-page gel, since my samples flow over to the other wells. It is a technique fault and I was wondering how I can improve that., I am in a very poor lab (at a very prestigious uni.) so I do not think they will buy speciliazed tips whatever for me
thanks

[danfive]

Gel loading tips are not very expensive, definitely look into them. Also gels have well capacity so check that they hold 10, 30, 50 microliters and adjust your sample volumes. As for technique just take your time and keep your eye on what you're doing, everyone masters it after a couple of trials.