Gel filtration/molecular sieving columns require more careful packing,
equilibration, and loading procedures than other types of
chromatography. In addition, column geometry is an important aspect:
you should have at least 30 units of column length for 1 unit length
in diameter (my own rough guess), if the purpose of purification is to
get your protein not in the excluded volume, as in desalting or
certain group separations. Flow rates for these columns are better
understood as linear flow rates (mm/unit time or cm/unit time) rather
than volumetric. If the manufacturer recommends 1 mm/min, then your
flow rate in ml/min is doing the math: 0.1 cm/min * col radius * pi.
Note that constant flow rates are essential, so your gravity flow
setup should not see the buffer level result in changes in pressure.
Your choice of fraction size (volume) will be on the difference
between the total column volume (Vt) and the void volume (Vo). If you
want that volume distributed in 50 fractions, then the fraction volume
is 2% of the difference (does not include fractions collected prior to
void).
The sample should ideally be 1/100th of the column volume, and applied
with the column flowing slightly (raise the effluent tubing upwards to
control flow) and after all eluent has entered the bed. Follow the
sample with eluent only after it has completely entered the bed; I
usually add a small amount of eluent, let it flow till all in, then
repeat a couple of more times, then establish regular flow. Another
problem is when the sample gets concentrated for gel filtration.
Often precipitates form, and these must be centrifuged out before
loading the sample; alternatively, if these precipitates are
worrisome or even contain your molecule, try re-solubilizing. Perhaps
a buffered saline will help. Or better, dialyze and concentrate in a
buffer with sufficient ionic strength (such as a buffered saline).
There are many dye-labeled (colored) protein standards designed nicely
for gel filtration that you can see coursing through the column, or
you can set up (unmarked) standards and follow the OD280/OD254 trace.
The dyes should move uniformly/symmetrically down the length of the
column. Any skew means the column has probably not been poured
properly, or that you have not applied the sample appropriately.
On 23 Apr 2002 15:08:25 -0000, Erin <enwarren at unity.ncsu.edu> wrote:
>So I have been having problems running my gel filtration column. It is a
>High Resolution column put out by Amersham Pharmacia. Before loading my
>protein I equilibrate the column with 3 column volumes of buffer. This
>buffer among other things contains 150mM salt per the instructions to
>eliminate nonspecific interactions of the protein with the column. Then I
>load my sample which has been concentrated down to under 1 mL and run the
>column at 1 ml/min. I collect about 2 mL fractions. When I run an SDS-PAGE
>gel of these fractions I see proteins of all molecular weights in each
>fraction, reflecting poor separation. It's as if I never put the protein
>sample through the column. I feel like it must be something I am doing
>wrong and not the column because at least one other member of my lab has
>used this column and achieved nice separation. Any thoughts on what the
>problem might be?
>
>Erin Warren
>>><http://www.biowww.net/forum/read.php?f=6&i=999&t=999>
>
