D:\612889668.doc
Wong Lab Protocols:
Coating Polyacrylamide with conjugated BSA:
(Preparation of coverslips adapted from Yu-Li Wang et al.)
Cover Slip Glass Activation & Polyacrylamide Preparation see: "Wong Lab substrate
prep protocol"
1.
2.
3.
Wash polyacrylamide gels in buffer with a pH 7
Activate the polyacrylamide surface with Sulfo-Sanpah according to previously
mentioned protocol
Prior to activation of surface: measure out desired BSA and conjugated BSA
dilutions.
notes:
 conjugated BSA (albumin from bovine serum, tetramethyl-rhodamine conjugate)
from molecular Probes: 3.6moles dye/mole
 molecular weight for BSA = 66,000 daltons
 buffer with PBS pH 7.4
 store at 4C with addition of sodium axide (2mM)
 for longer storage keep at -20C
4.
5.
Stock solutions are stored in .5mg/ml aliquots for both conjugated and
unconjugated BSA
Desired concentration of BSA should be around (.045mg.ml -.1mg/ml)
Experiment:
Part 1 (Specific attachment): .1mg/ml final concentration
 React each gel with Sulfo- Sanpah x1 for 10 minutes
 Place 200 ul BSA solution on each gel allow to absorb for 6-12 hrs.
Percentage
BSA
0%
50%
75%
90%
Percentage
BSA*
100%
50%
25%
10%
Number of
Samples
3
3
3
3
volume
BSA
volume
BSA*
Volume
Hepes (pH 8.5)
D:\612889668.doc
Part 2 (Non-Specific Attachement):
 DO NOT activate surface with Sulfo Sanpah
Percentage
Percentage
Number of
volume
BSA
BSA*
Samples
BSA
0%
100%
3
50%
50%
3
75%
25%
3
90%
10%
3
volume
BSA*
Volume
Hepes (pH 8.5)