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PDI western blot - can't reduce the disulfide bonds (Nov/05/2009 )

hi,

Please help. i have an antibody from santa cruz sc-17222 that should detect a band at 55kDa for PDI. PDI is present in liver so i am trying to use it as the positive control tissue, but i get the strongest band at ~110kDa, another weaker one at ~72kDa and the weakest one at ~55kDa. I always get three bands, no matter how long i denature or whether i use DTT or mercapto in the loading buffer. The band that i want is always the faintest one too.

I have seen in some papers that people use iodoacetamide to block the formation of dimers, but i have never tried this before as i have not come across this problem before?

Thanks

rachel

-rachelg-

rachelg on Nov 6 2009, 09:13 AM said:

hi,

Please help. i have an antibody from santa cruz sc-17222 that should detect a band at 55kDa for PDI. PDI is present in liver so i am trying to use it as the positive control tissue, but i get the strongest band at ~110kDa, another weaker one at ~72kDa and the weakest one at ~55kDa. I always get three bands, no matter how long i denature or whether i use DTT or mercapto in the loading buffer. The band that i want is always the faintest one too.

I have seen in some papers that people use iodoacetamide to block the formation of dimers, but i have never tried this before as i have not come across this problem before?

Thanks

rachel


they might not be disulphide linked dimers!!! may be hydrophobic... try runing urea gel... might help.. not sure though..
the 72kDa one might be some association of some degraded part of the molecule with the monomer..
Best luck!!!

-Pradeep Iyer-

I don't know if it helps much but i did a western blot for PDI today and I used the MA3-019 antibody from Affinity Bioreagents and it worked excellent. There's just one strong single band. I use PDI as a loading control for membrane fractions from liver.

-tea-test-

Pradeep Iyer on Nov 6 2009, 12:27 AM said:

rachelg on Nov 6 2009, 09:13 AM said:

hi,

Please help. i have an antibody from santa cruz sc-17222 that should detect a band at 55kDa for PDI. PDI is present in liver so i am trying to use it as the positive control tissue, but i get the strongest band at ~110kDa, another weaker one at ~72kDa and the weakest one at ~55kDa. I always get three bands, no matter how long i denature or whether i use DTT or mercapto in the loading buffer. The band that i want is always the faintest one too.

I have seen in some papers that people use iodoacetamide to block the formation of dimers, but i have never tried this before as i have not come across this problem before?

Thanks

rachel


they might not be disulphide linked dimers!!! may be hydrophobic... try runing urea gel... might help.. not sure though..
the 72kDa one might be some association of some degraded part of the molecule with the monomer..
Best luck!!!


we use homogenous20 gels for Phast Transfer System, can i treat the samples with urea prior to running the gel instead? I will see if they have a urea gel in their catalogue too.

Thanks :P

rachel

-rachelg-

tea-test on Nov 6 2009, 10:07 AM said:

I don't know if it helps much but i did a western blot for PDI today and I used the MA3-019 antibody from Affinity Bioreagents and it worked excellent. There's just one strong single band. I use PDI as a loading control for membrane fractions from liver.


do you just use a standard sds protein extraction buffer and heating for 5mins at 95-100 then spin for 10mins and take off supernatant?

Thanks

rachel

-rachelg-

Hi, I used the Fermentas ProteoJet membrane extraction kit and no boiling, just 30min at 37C before application.

-tea-test-