Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 14)

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Section 6
Protein Preparation
Bring all buffers to room temperature prior to use. We recommend setting
up as many as ten protein coupling reactions at one time using various
amounts of protein in order to determine the optimal coupling conditions
for your protein.
1. a) If your protein sample is not free of sodium azide, BSA, glycine,
Tris, or amine containing additives, proceed to step 2 and follow
the buffer exchange procedure using Micro Bio-Spin 6 Tris
chromatography columns. Alternatively, dialyze overnight against
PBS, pH 7.4.
Or
b) If your protein sample is already suspended in PBS, pH 7.4, and
is free of sodium azide, BSA, glycine, Tris, or amine-containing
additives, determine the protein concentration with the Bio-Rad
DC
protein assay kit or any other protein assay of choice.
2. Use one Micro Bio-Spin 6 Tris chromatography column for each
different protein requiring buffer exchange with PBS, pH 7.4. The
exclusion limit for the Bio-Gel
® P-6 gel contained in the columns is
6,000 Da.
Note: Up to 20% of the protein can be lost during this buffer
exchange procedure. For further instructions, refer to the manual
provided with the columns. The manual can also be found on the
Bio-Rad website (www.bio-rad.com).
3. Invert the column sharply several times to resuspend the settled gel
and remove any bubbles. Snap off the tip and place the column in a
2 ml microcentrifuge tube (included with the chromatography
columns).
4. Centrifuge the column for 2 min in a microcentrifuge at 1,000 x g to
remove the remaining packing buffer. Discard the buffer.