These notes are not designed to teach the theory of gel filtration, nor to
be a detailed primer on the use of these columns. This is just to provide some
ideas about how to perform this function relatively efficiently--since it is a
common method in every conjugation protocol (most conjugations will require
between 1 and 3 columns per antibody).
We use either PD-10 or Presto columns for our separations, depending on the
volume of sample. For sample volumes under 0.5 ml or over 2.5 ml, consider
pouring your own columns.
In general, you will end up collecting approximately 0.5 ml more than you
load on the column (for larger volumes, the increase can be as much as 25% of
the starting volume). Try to collect less total volume rather than all of the
protein; the last 10% of the protein coming off the column probably adds 30% or
more of the volume to the collection.
There are three easy alternatives for column separation of proteins which do
not have visible coloration. We have now adopted the third as the easiest and
most convenient.
As soon as you have loaded the protein onto the column, start collecting
0.25 ml fractions. Use Eppendorf tubes lined up in a rack: place the first tube
under the column, and add 0.25 ml of buffer onto the top of the column. When
the column stops draining, move the rack such that the next tube is under the
column; add another 0.25 ml. Continue collecting fractions; generally, you will
need to collect between 8 and 16.
Method 1: Spectrophotometric.
Measure the Absorbance at 280 nm of each fraction. Do not dilute the fractions;
use a 300 microliter cuvette. After measuring the absorbance, transfer the
fraction from the cuvette back into the eppendorf tube. Combine the fractions
which contain approximately 90% of the total absorbance.
Method 2: Biochemical. On a piece of
wax paper, place successive 2 microliter drops of Bradford Reagent (see below)
about 1 cm apart--one drop for each fraction. Mix each fraction thoroughly;
remove 6 microliters of each fraction and mix with a 2 microliter drop. The
protein will turn the drop blue; combine the fractions which have the bluest
color compared to the buffer.
Method 3: Tracer addition. Before
loading a nonvisible protein on a column, mix it with 25 microliters per ml of
antibody of 50 mg/ml Blue Dextran (see below). Mix thoroughly, and apply to the
column. Blue Dextran has a large molecular weight and will mark the position of
the antibody. Then follow the instructions below for "Separation of
visible proteins." We have not found Blue Dextran to interfere with any of
the conjugation reactions; however, if you encounter problems with your
conjugation, you may want to try leaving out the Blue Dextran and using Methods
1 or 2 for the separation.
When you have a protein (conjugate) that is easily visible to the eye (for
instance, phycobiliprotein conjugates, FITC conjugates, etc.), then column
separation is very easy. For nonvisible proteins, you may add Blue Dextran (see
Method 3 above) to mark the volume to be collected. When you have a clearly
colored protein solution, then you will just "collect the color."
Apply the protein to the column; once it has drained to the top of the column,
add buffer to the top. Watch the tip (bottom) of the column carefully; as soon
as color appears in the drops, start collecting the eluate (it is convenient to
collect this into a Eppendorf tube, if the starting volume was less than 0.75
ml, or a 3.5 ml screw-cap tube otherwise). Keep collecting eluate until the
color of the drops off the column is very pale. Don't try to keep collecting
until the drops are clear; the last 10% of the protein will add at least 30%
volume to the eluate... try to keep the conjugates as concentrated as possible!
A rule of thumb is that you will collect about 0.5 to 1 ml more than the
initial volume; this may vary depending on the amount you load and the type of
column you are using.
For 1.25ml to 2.5ml sample volumes: PD-10 (Sephadex G-25M), Pharmacia
Biotech, catalog No. 17-0851-01.
For 0.5 to 1.5ml sample volumnes: KwikSep dextran desalting columns, Pierce,
catalog No. 43232.
Bradford Reagent: Sigma catalog number B6916 (500 ml). Use undiluted as
described above.
Blue Dextran (25x stock): Sigma Catalog Number D5751. Make 50 mg/mL in
distilled water with supplemented with 0.1% (w/v) NaN3 (azide).