Protein Lysate and blotting problems - (May/22/2006 )

I am having the most bizarre results with my westerns!

1. Basically the loaded amount of lysate does not show equal loading despite re-quantification and getting exactly same result! I know that there is no degradation as even the low expression proteins come thru and are good bands, its just after actin, i can see that the loading is not equal at all therefore defeating the purpose of quantifying!

2. Today the problem was abit more complicated, one of my sample appeared very weak on a number of Ab staining blots, I was expecting a weak actin band but the actin seemed absolutely fine and infact there was more of it than in the other samples!!! what's going on?

3. Does anyone have a recipe for a good lysis buffer (plus inhibitors) I have bee using the TGN buffer with about 5 inhibitors , I am not sure what to change with the westerns as I have tried many different approaches but have been getting same horrible results! basicall all my cell pellets are in liquid N2 , i do all the lysis on ice, basically I observe all the important things yet this has been an ongoing problem, I am working with Hodgkin cell lines if this is helpful

If anyone has a suggestion or a brand new protocol for sensitive cell lines I would be grateful for them. many thanks

Hi

About the problem with protein quantification:
the problem might be due to the presence of components in your lysis buffer.
For eg. triton X-100 is a well known detergent used in common cell lysis buffer like RIPA buffer, but the disadvantage it interferes with reading in spectrophotometer. Therefore it is important to add the same amount of buffer to your blanck and to all your protein standards, this way the effect of the buffer will be introduced in all samples. Doesn't mean that this will completely solve the problem but it will certainly improve.
NP-40 is not so problematic as Triton X-100

About the problem of low signal:
It can be that your cells were not properly lysed and therefore you lost a significant amount of it that precipitated together with cell debri.
In case your protein is nuclear or membrane bound protein is more difficult to extract it. Lysis is a crucial step. I advise you to first check what is the best buffer.
Try several and see which ones gives better yield of your protein.

Good luck

many thanks, will definitely consider these points next time