Hi I am trying to express my protein R in tobacco leaves and do a CoIP, but I can't detect the protein R in CoIP input.
I took the same tissue and added 8M Urea plus LDS protein buffer, protein R shows as a distinct band at the correct size. But when I grind the tissue and put in CoIP lysis buffer, after centrifugation, I put the input in LDS, I couldn't detect my protein, I only see a smear.
Does anyone know what went wrong?
Hi
a smear usually indicates degradation. Do you use ice-cold buffers and also protease inhibitor (cocktail)?
Best
Sebastian
hm difficult. Are you heating your samples on 95° prior to SDS PAGE? This is most likely a problem of components in the IP-buffer. High content of PEG can cause such smears (my experience) and IP Kits often contain high amounts of PEGs. Maybe precipitate your sample before SDS-PAGE (Acetone or Methanol/chloroform), then resolubilize in LDS by boiling and see what happens. Methanol/Chloroform is usually better for depletion of detergents.
Best and good luck!
Sebastian Hoernstein Hi Sebastian, thanks very much for you answer! I suspect it is also something wrong with the IP-buffer. I am using the SII buffer, which contains the following:
100 mM Na-Phosphate, pH 8.0 150 mM NaCl 5 mM EDTA 5 mM EGTA 0.1% Triton X-100
I also added PIC, Bortezomib, and NaF before use.
Do you see anything wrong in my lysis buffer?
One more weird thing, my protein R is tagged with HA and I have another protein X tagged with Flag. When I detect the same sample, I can detect protein X just fine in the IP input, however, the protein R with HA-antibody gives me a giant smear. I asked my lab mates and people nearby, no one has any explanation.
I am attaching two blot pictures. These two are from the same sample, the clear one is 8M urea + LDS, the smeary one is after CoIP lysis buffer.
I tried different things, like switching the buffers, switch antibody, adjusting loading amount, adjusting antibody concentration, optimizing protein lysate preparation steps, I am really running out of ideas and this has been bothering me for more than a year.
Please offer more insights, many thanks!
Hi Yuzhao,
sorry for the delayed response. Well it looks really difficult. Did you try to precipitate your sample before loading on a gel? This might really be a good idea if it´s really (and that´s what it looks like) the IP buffer. Then you can dissolve your sample in pure lämmli and boil it prior to SDS-PAGE. However, to be honest, I´m also running a little bit out of ideas.
Good luck!
best
Sebastian
Sebastian Hoernstein Hi Sebastian, thanks a lot for your response. I think I actually solved this problem. I changed our PIC to the cOmplete mini tablet from Roche and I saw the band. It seems like our PIC was not working and my protein of interest was degraded. And the HA antibody has a very high background noise for tobacco extracts in general. So, when the protein of interest was degraded to a certain level, I would end up seeing the smear pattern, which is all background noise from the HA antibody staining. I also tried just the LDS and it works.
Yuzhao Hu Just to ask, which PIC did you use before changing to cOmplete?
Best
Sebastian
Sebastian Hoernstein My PI ordered AEBSF, E-64, Bestain, Pepstatin, Leupeptin and 1, 10-Phenanthroline separately as powder and mixed them together manually and dissolved them in DMSO, which resulted in the 100X PIC I used. He made two batches and I think the old batch he made worked while the new batch was bad, and that is why I have been getting this smear/degradation for a year. And the time when he made the new batch roughly match with the time I started to see the smear/degradation.
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