High background in western blot - (Nov/20/2008 )
I've been having some problems with my Westerns. I created a stable cell line with an overexpressed gene (with an HA tag). I grow my cells in 100 mm dishes and lyse them with 1 ml RIPA buffer then do freeze-thaw three times and spin at 14000g x 15 min. I don't get much in terms of a pellet but I "save" my supernatant in any case. Biorad assays show around 0.5-0.6 mg/ml protein concentration. I load about 25 ul sample on my 10% gel. Run gel and semi-dry transfer. Block with 5% BSA in TBST. 2 hr. Primary Ab 1:500 (diluted in TBST), Wash, Secondary Ab (diluted in TBST): 1:750, CL, and expose. I have a feeling my secondary ab is not dilute enough but I'm not sure what concentration to use. Is it possible to re-use my membrane (re-do antibody incubations) after I've gone through the procedure mentioned above? Any suggestions would be great. I have not had great luck with the few Westerns I've done....
Your second antibody seems indeed to be the problem. Try to do an experiment with just positiv and negativ controls for an dilution series with the second one, ranging from 1:500 up to 1:10.000. Normally, the supplier gives instructions on the starting dilution, normally around 1:2000 or 1:5000. How long do you block? How long do you incubate in 2. Ab? 1h for each is enough.
You can re-use your membrane, but you have to strip the old antibodies and ECL first with stripping solution (low pH oder ß-ME), after this you can reprobe the membrane starting with blocking, then normal routine. Oh, that means, if you are using PVDF-membranes. I think, this is not possible with nitrocellulose, but I am not sure.
You may also want to try different blocking solutions. I haven't had to change much from BSA, but some people swear by blocking with milk. Also your secondary ratio may be wrong, i have never had to do a secondary dilution more concentrated then 1:2000; often i've had to back it off to help bring down the background. Also as suggested perhaps your secondary time is off, don't put secondaries on for more then 2 hours. 1 hour is often sufficient.
Try adding BSA to your antibody dilutions as well. We use 1% BSA or milk in the primary Ab and 0.2% BSA or milk in our secondary Ab. The milk or BSA should be made fresh when it is to be added to antibody dilutions. This should reduce background binding.
In our experience, milk is a better and more cost-afficient blocking agent. However, milk can in many cases inhibit binding of phospho antibodies. Use BSA when hybridising with phospho Ab.
I've been having some problems with my Westerns. I created a stable cell line with an overexpressed gene (with an HA tag). I grow my cells in 100 mm dishes and lyse them with 1 ml RIPA buffer then do freeze-thaw three times and spin at 14000g x 15 min. I don't get much in terms of a pellet but I "save" my supernatant in any case.
I don't get it . your protein of interest should be in the pellet ?
I have also the feeling that your secondary antibody is not enough diluted.
Usually you dilute it from 1:5 000 up to 1:100 000.
do not incubate too long with secondary antibody. 1 hour at room temperature is enough.
after three washes with TBST, wash twice with TBS before CL to get rid of tween.