Hi,

I'm trying to purify a recombinant polypeptide (43 residues long) using the baculovirus expression system. I've been successful with this system isolating and analyzing full size protein (60-90KD) but this is my first attempt using the system for a polypeptide.

First, is a polypeptide this small possible to construct + isolate using the baculovirus expression system?

Second, anyone know any literature or have experience that can offer advice?

Third, If this isn't possible, whats the best way to produce a polypeptide this small, synthetically? using another system?

Thanks in advance.

Hi,

I would like to study extracellular matrix secreted by cultured cells. The problem is how to remove the cells but still keep the ECM as intact as possible. After removing the cells I would like to plate new cells and study their adhesion etc. on to the plate.

If someone knows an articel or has experience on the matter I would appreciate the help.

Hi all,

Does anyone can give me some hints on why i cant see one of the proteins in the native gel? I can see some other proteins im running on it but not the smaller (acording to SDSPAGE) I've tried to added after 30 mins in case it was going out but without success.
working pH is 8.8 in a 7.5% gel

Thanks!!

Hello,

1. Once the PAGE is prepared, how long can it be stored and under what conditions? For instance, if I prepare the gel on Friday afternoon, can I put the set gel in tank buffer and then store it in the fridge till Monday? Or would simply wrapping the gels in mQ H2O soaked paper towels work just as well?

2. I'm having trouble with our Santa Cruz alpha-tubulin antibody (SC-8035). It doesn't work well---its temperamental, sometimes it works and other times not and we don't know why. Anyone else having these problems? Found a solution? Alternatively, can one suggest another anti-house-keeping antibody which can be used as a loading control but also has multispecies activity. Ideally, I'm looking for something active against rat, goldfish and zebrafish. An alternative alpha-tubulin antibody would be most welcome.

p38 has been suggested and this would be great as my target proteins are all much larger than 38kDa but does it have multispecies activity?

Thanks

6xHis-Tag protein purification under denaturing conditions.
General Protocol is:
Sonicate in tris/NaCl - spin - Sonicate pellet in 2M Urea - Spin - Incubate pellet for hours in 6M GuHCl/20mM Tris/500mM NaCl/1mM b-mercaptoethanol pH 8 - spin - Filter supernatant - run filtered supernatant on Ni-NTA.

What I have been seeing for a while and just dealing with it is a very very low affinity for Ni_NTA, somewhere around 50-75mM imidazole elution.

To answer the obvious questions : yes, I have extensively evaluated the solubility and 6M GuHCl is required and the pi of the protein is around 5 and my Ni-NTA is equilibrated in the pH 8 GuHCl buffer mentioned above. According to sequencing following cloning there is a 6xHIS tag

The only thing I can think of lately is that the sample is of course still has a level of viscosity. I mean, it's not very high but still present.

Could it be that or is there just something else I am missing? I mean this is a very standard protocol.
The only thing I am not doing that many are now is on-column refolding. I wish I could but I am actually working on several proteins of the same family and some just love to aggregate on the column - I have tried.

Any ideas, opinions, comments, questions?
Thank you very much