Hi there everyone,
What does it mean when you have proteins eluting in almost the whole
column volume of S200?
I ran a gel with fractions from 8ml to 20ml and saw band for my
protein all throughout.
Judging peaks on chromatogram is not useful as it doesn't have any
aromatic rings.
Che
It possibly means:
1. Your sample application volume was too large
2. Total protein quantity was too large
3. Fraction size was too large
4. Sample was too crude (GEC is a finishing step, not a starting step for
protein purification.
5. Sample was too concentrated/viscous
6. Flow rate was too fast
Following on from Roger's fine suggestions:
8. Your column is knackered. Can you see fine lines or cracks in the
column? Good packing is v.important for SEC columns.
HTH,
Dave
David C. Briggs PhD
Father, Structural Biologist and Sceptic
Hi, David,
What is the common cause of knackered SEC column? Will equilibrizing a
buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause
the problem. There was no problem just after packing the column.
Nian
On Sun, Aug 28, 2011 at 9:24 AM, David Briggs wrote:
> Following on f
Hi Nian,
It can be a number of things - but typically, air getting into the
column, or a leaky seal somewhere letting it dry out.
Switching from 150mM NaCl to 20% EtOH directly shouldn't cause a problem.
The reason I suggest it is that I have had the same situation as
described in the original p
worst-case scenario for crystallization purposes:
9. Your protein runs as a mixture of monomers, dimers, trimers,
whatever-mers...
Filip
On Sun, Aug 28, 2011 at 7:24 AM, David Briggs wrote:
> Following on from Roger's fine suggestions:
>
> 8. Your column is knackered. Can you see fine lines or
Testing for cracks in the column is quite easy. Take whichever standard you
happen to have (lisozyme, GST, etc) and do a run. If it comes out in a
single peak the problem is with your prep. If it comes out throughout the
run the problem is with your column.
Mario Sanches
On Sun, Aug 28, 2011 at 5
Nian,
Before you dump the column, clean it and run some protein standards on
it. If everything looks OK, run a small sample of your protein
again. If it behaves the same way, then you may have a protein with
hydrophobic patches. Anomalous binding to and elution from polymeric
SEC media
Thanks everyone for your response.
The most likely answer to my problem is protein overloaded onto the
column. I pushed my protein concentration further down to 0.5ml
instead of the usual method, which to run multiple times on SEC.
Adding NaCl in the buffer may also help, as it seems that t
I'm glad this worked out well for you. For
conducting molecular sizing determinations with a calibrated GEC
column, I typically use 100-500 uL injections of approximately 1
mg/mL or so of purified protein on a 1.6 x 60 cm column. The
limited protein quantity will
Thanks, David. I believe the leaked column was probably my case,
although I didn't see any visible leakage I re-tightened connections
anyway. But the weird thing was that after I equilibrized the column
for a while, the dry patches and cracks disappeared. Everything
returned to normal even a standa
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