Smeary western blots of cell lysates - western of a HMW membrane protein (Jun/24/2008 )
Dear All,
I am having problems with running western analysis on an integrin protein. I lyse my cells in RIPA buffer. The RIPA buffer includes Benzonase to get rid of the DNA. After lysis i mix the lysates in SDS loading buffer without reducing agents. I then load the lysates and resolve them on a 7.5 % SDS gel. I run two gels in parellel and then transfer to PVDF membranes. The Size markers transfer well to the membranes. I then block and bot the antibodies. One membrane is blotted against alpha7 integrin and the other is blotted against actin. Both result in smeary lanes. I cannot see any bands. I am loading a small amount of protien in the lanes ~ 1 microgram. I am not sure how to resolve this issue. Do you have any suggestions?
Will centrifuging the samples before loading help?
Thank you.
I am having problems with running western analysis on an integrin protein. I lyse my cells in RIPA buffer. The RIPA buffer includes Benzonase to get rid of the DNA. After lysis i mix the lysates in SDS loading buffer without reducing agents. I then load the lysates and resolve them on a 7.5 % SDS gel. I run two gels in parellel and then transfer to PVDF membranes. The Size markers transfer well to the membranes. I then block and bot the antibodies. One membrane is blotted against alpha7 integrin and the other is blotted against actin. Both result in smeary lanes. I cannot see any bands. I am loading a small amount of protien in the lanes ~ 1 microgram. I am not sure how to resolve this issue. Do you have any suggestions?
Will centrifuging the samples before loading help?
Thank you.
1. Add anywhere between 25 to 100 microg extract per well (if you are adding just 1 microg, there is your real problem).
2. Since your actin also does not work, I don't think it is a problem related to membrane protein, you need to troubleshoot western technique in general.
3. I would add reducing agent.
4. No boiling
5. Add sonication to the mix. It may increase the protein amount.
6. Use a positive control with alpha7 integrin overexpression, or a cell line with high expression of teh same.
7. Always centrifuge.
hth/
THank you for your suggestions
I use myotubes as controls for high integrin expression. This control is the last lane on the gel. This lane looks exactly like the other lanes on both gels. I am creating an expression system in E.coli and will be able to use this as a control later. Thank you.