8 replies to this topic

[doriskarloff60]

Hi, this is my first post so bare with me!

I've been trying to make some phospho-antibodies work for what seems like eternity without any success. I've optimised the IP for my protein of interest, increased the size of reactions so maximise the protein I can load onto my gel and used all the phosphatase and protease inhibitors when I lyse my cells.
With regard to the Western blot, I get good separation (as seen by Instant Blu staining), and transfer works well (as seen my Panceau S staining), I'm currently using nitrocellulose, and understand PVDF might be beneficial so I will try that.

But I just think that the phospho-site is being lost during the assay, if anyone has any ideas on how I can retain this phospho-site (been identified from Mass Spec) any help would be very much appreciated!

Thanks,

[knuf]

What do you mean that the phosphosite is being lost? How are you seeing that? Do you think that it is the assay or the detection that is failing? One thing to be aware of is that many phospho-specific antibodies are not compatible with milk blocking buffer because milk proteins (particularly casein) are phospho-rich proteins and thus, can quench your antibody and also contribute to background noise.

[doriskarloff60]

Hi, thanks for the reply.
The phospho-site has been detected by mass spec, yet I can't find it with these specific phospho-antibodies. I've already been using 3% BSA to block and incubate my antibodies in.
I'm not sure what's failing, it's hard to know if it's the antibodies that don't work, or there just isn't enough of the phospho-site preserved to be detected?

[knuf]

doriskarloff60, on 04 March 2011 - 01:16 AM, said:

Hi, thanks for the reply.
The phospho-site has been detected by mass spec, yet I can't find it with these specific phospho-antibodies. I've already been using 3% BSA to block and incubate my antibodies in.
I'm not sure what's failing, it's hard to know if it's the antibodies that don't work, or there just isn't enough of the phospho-site preserved to be detected?

Do you have any idea about the spectral counts of the phospho-site? Could it be that yes, there is some phosphorylation, but the relative amounts are below the level of detection for the antibody?

[doriskarloff60]

I think that is what is happening, that the relative amounts are below detection levels. Is there any way to boost the levels of phosphorylation or to decrease the levels required for detection?

[knuf]

Pierce sells phospho-enrichment kits...I've never used it but you could check it out maybe. It is product number 90003.

[Chelo]

doriskarloff60, on 03 March 2011 - 02:01 AM, said:

Hi, this is my first post so bare with me!

I've been trying to make some phospho-antibodies work for what seems like eternity without any success. I've optimised the IP for my protein of interest, increased the size of reactions so maximise the protein I can load onto my gel and used all the phosphatase and protease inhibitors when I lyse my cells.
With regard to the Western blot, I get good separation (as seen by Instant Blu staining), and transfer works well (as seen my Panceau S staining), I'm currently using nitrocellulose, and understand PVDF might be beneficial so I will try that.

But I just think that the phospho-site is being lost during the assay, if anyone has any ideas on how I can retain this phospho-site (been identified from Mass Spec) any help would be very much appreciated!

Thanks,

Are you sure the antibody works well? Any positive control in your WB? Actually, many phospho-specific antibodies in the market do not work at all!

[doriskarloff60]

We've no idea if the antibody works, thats the problem. We've had two designed for the peptide by eurogentec, but neither of them are producing a band.

[HBImolbiol]

doriskarloff60, on 11 March 2011 - 05:43 AM, said:

We've no idea if the antibody works, thats the problem. We've had two designed for the peptide by eurogentec, but neither of them are producing a band.

Almost all generalized anti-phosphoserine and phosphothereonine antibodies don't work, despite what the manufacturer claims.  Unless you have made one specifically to the phosphorylated antigen in your protein of interest.  Anti-phosphotyrosine antibodies tend to be much better.

To increase your chances, also consider including a cocktail of phosphatase inhibitors in the lysis buffer (ie 10 mM pyrophosphate, 10 mM NaF, 1 mM vanadate).

Edited by HBImolbiol, 24 March 2011 - 12:35 PM.