1 reply to this topic

[JennyHara]

Dear All Western Blot Experts,

I've been trying to detect pattern of phosphotyrosine proteins ranging from 210kDa to 37 kDa for over a month now but i always failed to get proteins from higher molecular weight approximately 210 kDa.

Now i'm using cell signal's phosphotyrosine antibody and two types of gels:
1. 7.5% gel
2. gradient gel 5-20% gel (both purchased from biorad)

Transfer buffer containing 5% methanol and transfer at 15V for 45 mins in semi-dry condition (We only have semi-dry machine)

Most of the time, the positve control cells transfering at this condition a nice bands patterns could be detected. However, in the experimental cells, the amount of phosphorylated maybe small that none of them appear on the PVDF membrane. In addition, the background levels are extremely high.

I'd checked on gel and observing that high molecular weights are still on the gel. I tried to increase time to 55 mins but it wont help and longer exposuring time, i'm afraid that the small size proteins will also be undetectable.


Please help!!! ....

And Thank you very much for all the suggestions

[PiDC]

It seems a problem with the transfer. I have made many phospho-tyrosine and transfer with a wet machine without methanol (I don't know if this is better or not, but I only have wet machine )...
try the transfer for longer times, or overnight, and check again for the transfer.

Other problem could be PVDF. For example, for the 4G10 antibody (anti-phospho-tyrosine), PVDF gives a dark background and nitrocellulose is better.

Maybe could help treatment of your cells with pervanadate as a positive control for phosphorylation. It gives a really high signal, so you could see your band of interest phosphorylated with this treatment.

good luck!!

JennyHara, on Aug 16 2009, 03:04 PM, said:

Dear All Western Blot Experts,

I've been trying to detect pattern of phosphotyrosine proteins ranging from 210kDa to 37 kDa for over a month now but i always failed to get proteins from higher molecular weight approximately 210 kDa.

Now i'm using cell signal's phosphotyrosine antibody and two types of gels:
1. 7.5% gel
2. gradient gel 5-20% gel (both purchased from biorad)

Transfer buffer containing 5% methanol and transfer at 15V for 45 mins in semi-dry condition (We only have semi-dry machine)

Most of the time, the positve control cells transfering at this condition a nice bands patterns could be detected. However, in the experimental cells, the amount of phosphorylated maybe small that none of them appear on the PVDF membrane. In addition, the background levels are extremely high.

I'd checked on gel and observing that high molecular weights are still on the gel. I tried to increase time to 55 mins but it wont help and longer exposuring time, i'm afraid that the small size proteins will also be undetectable.


Please help!!! ....

And Thank you very much for all the suggestions