Western Blotting for Phosphorylated Proteins

[sciencemandan]

I am trying to detect a phosphorylated protein using a western (this is my first time working with phosphorylated proteins). I have an antibody that has been shown to work quite well the literature (purchased from Cell Signaling). I am having issues with getting consistent results, though.

Here's the protocol I am using:
-Harvest cells in 2X Laemmli buffer and store at -20°C.
-When ready to run SDS-PAGE, remove samples from the freezer and boil them for 20 minutes, then load 20-25 microliters onto a 12% gel (the protein is around 50 kDa). Run the gel and electroblot onto a PVDF membrane.
-Allow the membrane to dry, then block in 5% milk for an hour at room temperature.
-Wash the membrane twice quickly with PBS/0.1% Tween-20 to get the milk off of the membrane.
-Incubate in the primary antibody (1:1000 dilution) in 5% BSA in TBST overnight at 4°C.
-Wash twice quickly with PBS/0.1% Tween-20, then do two 10-minute washes.
-Incubate in the secondary antibody (1:30000 dilution) in 5% milk for 1 hour at room temperature.
-Wash twice quickly with PBS/0.1% Tween-20, then do two 10-minute washes.
-Develop.

The main thing I am thinking of switching out is the buffer I am using to lyse the cells. It should in theory denature everything and remove phosphatase activity, but I'm wondering if I'm still getting some phosphatase activity, which could be causing my problems. I have not tried to add phosphatase inhibitors, but I see no reason to do so if the Laemmli buffer denatures everything.

So, what should I try now?

[admin]

Hi Sciencemandan
welcome to the site

I have some experience with these, usually the best thing to do is lyse with loading buffer the entire well you grew your cells on, keep everything cold but use fresh.

Also dont forget you need to use phosphatase inhibitors! I forget the names of these its been a while but we add them into the lysis buffer, and other buffers like dialysis. They really do make a difference trust me.

cheers

[sciencemandan]

Originally Posted by admin

Hi Sciencemandan
welcome to the site

I have some experience with these, usually the best thing to do is lyse with loading buffer the entire well you grew your cells on, keep everything cold but use fresh.

Also dont forget you need to use phosphatase inhibitors! I forget the names of these its been a while but we add them into the lysis buffer, and other buffers like dialysis.

cheers

Thanks for the quick response!

I am indeed lysing with the loading (Laemmli) buffer, then transferring to microcentrifuge tubes and storing in the -20°C freezer until I need them.

About the phosphatase inhibitors: do I still need these even though the Laemmli buffer should in theory denature any phosphatases? I didn't think I'd need to add them, but perhaps I should just to make sure.

The protein I'm working with is only phosphorylated on two specific serine residues, so I should just need a serine phosphatase inhibitor (like NaF), right?

Thanks again for the help.

[sciencemandan]

Oh, also, do I need to add phosphatase inhibitors to the blocking buffer and/or the antibody mixtures just to be safe?

[sciencemandan]

Whoops...for some reason my first post didn't actually post anything. The main question I had is whether or not I really needed phosphatase inhibitors since I am using Laemmli buffer (which should in theory denature everything, including phosphatases).

[pigfarmer]

Originally Posted by sciencemandan

Whoops...for some reason my first post didn't actually post anything. The main question I had is whether or not I really needed phosphatase inhibitors since I am using Laemmli buffer (which should in theory denature everything, including phosphatases).

Can't hurt to put phosphatase inhibitor in. When you say you are getting inconsistent results, do you see bands with the antibody for the non phospho form?

[sciencemandan]

I'm just getting a lot of background and, when I am able to see bands, they are broken up and look pretty bad.

I have blotted with the non-phospho form and have no problems. Based on the experiment I am running, I definitely should be seeing the phospho form, so perhaps I should just give the phosphatase inhibitors a try.

[pigfarmer]

Originally Posted by sciencemandan

I'm just getting a lot of background and, when I am able to see bands, they are broken up and look pretty bad.

I have blotted with the non-phospho form and have no problems. Based on the experiment I am running, I definitely should be seeing the phospho form, so perhaps I should just give the phosphatase inhibitors a try.

Yeah, I would definately add the pptase inhibitors, can't hurt. But you have gotten good signal from the Ab? Maybe you just need to optimize the antibody, since it is new, right?

[sciencemandan]

Originally Posted by pigfarmer

Yeah, I would definately add the pptase inhibitors, can't hurt. But you have gotten good signal from the Ab? Maybe you just need to optimize the antibody, since it is new, right?

Yeah, it is new. I have tried running it at several different concentrations, and I found one that gives a decent signal. I'm going to try phosphatase inhibitors and see what happens.

[pigfarmer]

Good luck!