Inmost sun

Hello everybody,

I am having problems with protein quantification. I have my proteins resuspended in 0,2M Glycine pH 2.5 buffer. So far as I know, this buffer is incompatible with Bradford, Lowry and BCA assays (these methods are only compatible up to 0,1M Glycine). Does anyone know another method to measure my proteins and/or do you have any suggestions?

Thanks in advance,
Cheers,
Andreia

I'm not completely sure why you're looking at transgenic models for intracellular calcium measurements.  Fura-2 is a standard ionophore for measuring calcium levels in cells in response to chemical stimuli.  You might also look at Cameleon, a CamKII-GFP hybrid protein that can be transfected or transduced into cells to visualize calcium concentration.

Hi Everyone,

  This is my second post in this section.
  Attached herewith is the pic of my SDS-Page of protein purification that I've done.
  The 8,9 and 10 column were the protein that I need to purify.
  Based from the bands that appear, I would like to confirm whether it is pure or not. Any advice,suggestion and ideas are welcome.
  Thank you.

Hi guys,
I've been asked to source all the equipment and reagents needed to set up western blot in the lab, starting from scratch (all we have is a Nupage electrophoresis tank).  I know my preferences for transfer system, membranes, reagents....  but I'm having doubts on the developing method. In the past I've always used ECL and x-ray films, but I know more and more labs these days favour other chemiluminescent reagents and imaging as you get a better control on exposure and a better dynamic range.

So here's my question.  

What method do you use to develop a western?    


Please share your opinions  

Thanks!  

Hello everyone.

I wanted to post this so that people that experiment with proteases could help. I'm trying to standardize a protocol for probing protein kinase C from cell lysates from QC and GM-95 cell lines.  The protocol that we are using is the following...


Lysate buffer

20 mM Tris HCl
0,5 mM EDTA
0,5 mM EGTA
0,3 mM Sucrose
0,5 mM PMSF

We expose the cells (grown in 50 ml bottles) to a toxin and wash it two times with PBS and for each time we lysate the cell culture adding a cockatil buffer and lysate buffer to each sample. We proceed to homogenize with a tuberculin syringe. Then a 15 minutes centrifugation at 8000 rpm at 4°C. Then we separate the supernatant from the pellet that will correspond to the nucleus fraction.
Then we centrifuge at 100000g for 1 hr at 4°C, and we separate the supernatant and the pellet. We add to the pellet 80 ul of lysis buffer with 1% of triton x-100.


We then proceed with the quantification which sometimes is really scarce of protein.

I'm wondering if anyone that works with protein kinase could help me find a better way to do my experiment.

Any ideas on how this occurs?  Been batting around some ideas but would like some input.

This solution

160 NaCl
2.5 KCl
1 CaCl2
2 MgCl2
10 HEPES
10 Glucose

has an osmolarity of about 320, how to calculate it from the molarity of the ionc composition.