weird query for Western Blotting - (Nov/25/2011 )

I am facing a real weird problem with western blotting of a trafficking molecule which is a 25 kDa GTPase located in recycling endosomes of the cell.
I load approx 10 ug protein usually for western blotting. the problem that i am facing is that the fresh lysates that are prepared in lysis buffer (Urea, tris base, CHAPS) does not giv me a band of interest, rather there are many non specific bands that are observed. But my old lysate (approx prepared 6 months before in the same lysis buffer) gives me a thick band of interest. This thing happens everytime i put up a western blot.
I have tried increasing and decreasing protein load, changing the secondary antibody, washing conditions, but the fresh lysates do not give me a band. I recently tried with another similar GTPase and it gives me a very prominant band without non specificity. Both the proteins are cytosolic.


The difficulty is that it is not possible for me to homogenize the samples as I have to transfect them in a 12 well plate and extract cells from maximum of 2 wells.. Hence i prefer adding lysis buffer to the cell pellet, overnight incubation at 4 degrees and homogenize using an insulin syringe, pelleting down at 10000 rpm for 45 mins and transferring the supernatant.

Another thing that I feel that my primary antibody works properly is that it detects the band in the old lysate but not in the fresh lysates or mock transfected cells.

The protocol that i follow is
10% gel run at 100 V, Transfer at 100 V for 1 hr 15 mins
Blocking with 2% blotto (ECL Advance kit, GE Amersham)
Primary antibody- 1:500 dilution, 4 degrees overnight
washing for 1.5 hrs with PBST containing 0.05% Tween 20
Secondary Antibody- 1:12000, 1 hr at room temperature
washing for 2.5 hrs with PBST containing 0.05% Tween 20
15 min washing with PBS
Detection with ECL Advance (Ge Amersham)

Help will be highly appreciated.

Hey,
It looks to me like you have an issue with the efficiency of protein lysis or SDS loading buffer treatment prior to running your WB.
You could add hot SDS loading buffer (containing β-Me and brought to 100oC) into your wells and scrape off the cells, instead of using a lysis buffer. Then use an insulin syringe to homogenize the lysate. For a 12 well plate you could use 100-150ul of hot SDS loading buffer per well. If it foams, spin it briefly in a centrifuge. Then just load it onto your gel. This way you can make sure that there is nothing wrong with your lysis buffer and your proteins are prepared properly for WB.

Thank you for your reply sir.. I ll try doing that..

Thank you once again