Homogenization of rat brains

[Protease]

Hi, I am currently working in a lab for the summer and the professor I have been working with wants to perform a 2D gel electrophoresis on rat brains. As an undergraduate, he has asked me to look up a procedure to perform 2d on rat brains which will assist him later on in his experiments, while it will help me get a good grade for coop/recommedation. I did find a procedure to homogenize rat brains but I am unsure of the amounts to use. I plan to use 0.01g of rat brain but in the article it says : Homogenization was performed in (Sol B) was: 7 M urea, 2 M thiourea, 40 mM Tris, 3 mM tributylphosphine,2% CHAPS, 1% Pharmalytes 3.5–10. How does one figure out how much to use for 0.01g of rat brain? I read through the article and no where does it mention how much to use (ul wise) to get good results when doing a 2d gel. Also, if anyone has a procedure of their own could post it here. The article I am using is Proteomic analysis of rat brain tissue: Comparison of protocols for two-dimensional gel electrophoresis

analysis based on different solubilizing agents. Any assistance would be helpful. Thank you for your time.

 

Regards

[CharonY]

In short: it does not matter. Generally you try to use the smallest volume possible in a 2D-compatible buffer that gives a good homogenate. As long as the buffer is 2D-compatible you can always dilute it to the desired amount (after cleanup). If you start with too much solubilization buffer you will need to add a concentration step (e.g. by precipitation or with MWCO filters). The solubilization buffer described in the OP has essentially the same composition as a normal IEF-buffer (with modification for membrane and other insoluble proteins) from what I can see. So there should be no problem there.

If you have no guidelines how much protein will be released with you homogenization procedure just use a small amount and work you way up.

[Protease]

In my lab the 7 M urea and 2M thiorea, CHAPS are all solids what would be a good base to use as a final volume liquid. In the paper they use Solution B which is composed of 7 M urea ..etc but I just have the chemicals in solid form and I was thinking either ddH20 or EDTA. Any recommendations?

 

Note :The second step after homogenization would be delipidation using ethanol/acetone

Edited July 1, 2009 by Protease

[CharonY]

In my lab the 7 M urea and 2M thiorea, CHAPS are all solids what would be a good base to use as a final volume liquid.

 

I am not sure what you mean. You have to create a buffer with the given concentrations (7 and 2 M respectively). Or are you not sure what molarity is? If that is the case I recommend to talk things through with your supervisor rather than try to incorporate suggestions from a forum. He/she is there to help you, but feedback from your side is absolutely crucial.

[Protease]

It's like a 2% agarose gel where I have the agarose but I am unsure if I need 100mL of EDTA or TAE to make it (solvent). I have all those chemicals but in solid form(CHAPS, Tris,Urea) and I want a solvent for those chemicals I was thinking either ddh20 or EDTA. Yet in that paper I listed above they have a ready made SolB (liquid) but in my case of I have to make a solution from solids.

[CharonY]

Unless otherwise noted the solvent is normally water.

Do not take it as a criticism, but from what you described it appears that you do not have much expertise in creating buffers and suchalikes. I would really recommend you to bring that up with your supervisor to avoid more problems down the road. Believe me, making buffers is the simplest part of the process. If the supervisor dumped you in the lab without actually knowing that you lack training, you won't benefit much from the lab work (even if it is only a summer course). Again, feedback is important.

[Protease]

Just an update :

 

I was being a dumbass ...sorry I just confused myself but thanks for the clarification and your patience. I did perform a 2d electrophoresis on the brain sample but loaded too much sample (I didn't think that silver staining would be as sensitive as it turned out to be). So far so good thanks again!