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Western blotting for ER - problems - (Aug/22/2006 )

I am currently doing Western blots for the presence of ER. I have tried numerous times with no results. I have optimised my antibody system using dot blot and gained positive clear results for that.
I am using a 3.5% stacking gel and 7.5% resolving gel, which should be correct for the 67kDa size of ER. I have gone through every aspect of my protocol and still cannot find why I am gaining no results, I use a prestained marker to ensure transfer, which transfers ok and develop using DAB tablets.
I have tried both NC and PVDF membrane, but am getting no positive results. Anybody got any ideas as I am running out of them.
Following transfer I stained my gel with coomassie blue and there was no protein in the gel, which, i assumed was because it had all transferred over. I transfer for 1hr at 4C, which is standard in most protocols. Due to the positive result with dot blot, could it be the transfer?
I am very to this technique so if anybody has any ideas what could be going wrong I would be very grateful.
Thanks

-Cree-

how much protein are you loading?
could you load more?
I never optimsed my antibody by dot blot, and I don't know if it's the best way to do it. (no discrepancy between background signal or specific signal), so I don't know what to tell you.

-Missele-

Did you ever do a ponceau staining of the blot? If you don't find any protein there, something is wrong with the amount of protein loaded or the transfer. Is there some kind of positive control you could load together with your samples on the gel and try to establish the blot with this? Would be helpful to optimise first and second antibody with it.

-biomaus-

QUOTE (Cree @ Aug 22 2006, 03:12 PM)
I am currently doing Western blots for the presence of ER. I have tried numerous times with no results. I have optimised my antibody system using dot blot and gained positive clear results for that.
I am using a 3.5% stacking gel and 7.5% resolving gel, which should be correct for the 67kDa size of ER. I have gone through every aspect of my protocol and still cannot find why I am gaining no results, I use a prestained marker to ensure transfer, which transfers ok and develop using DAB tablets.
I have tried both NC and PVDF membrane, but am getting no positive results. Anybody got any ideas as I am running out of them.
Following transfer I stained my gel with coomassie blue and there was no protein in the gel, which, i assumed was because it had all transferred over. I transfer for 1hr at 4C, which is standard in most protocols. Due to the positive result with dot blot, could it be the transfer?
I am very to this technique so if anybody has any ideas what could be going wrong I would be very grateful.
Thanks


I am loading 25ug of protein per lane and using lysate I collected from MCF-7 cells as a positive control.

-Cree-

biomaus is right, you should do a Ponceau red staining to check if everything is OK. You will be able to see if your bands are nice, and the amount of protein after transfer correct.

-Missele-

Use a 10 or 12% gel.

I do ER westerns very routinely and thats what I use. Transfer for 1 h at 100 V using an ice pack but at room temp.

good luck.

-Casper-

The protein leaving the gel if the coomasie is blank but the ponceau will show you if it's going onto the nitrocellulose and not into the buffer. There's no azide in your ab incubation buffers is there as that inhibits HRP and could cause problems with your detection. Have you tried using another antibody you know will work e.g. actin to see if it's down to the ER antibody or down to something else (transfer or the detection system)?

Ceri

-Ceri-

are you sure your membrane is on the correct side of the transfer sandwich?

is your membrane activated (pvdf)?

are you sure your protein hasn't blown through the membrane?

-mdfenko-