Bio-Rad Biotinylated Standards User Manual
Page 8
6.4 No Color in Sample, Good Color Development in
Standards
For problems with immune detection of the samples, consult Bio-Rad's
Immun-Blot assay kit instruction manual.
6.5 High Background
1.
Insufficient blocking prior to
Increase blocking step to 60
first antibody incubation.
minutes.
2.
Insufficient washing after avidin
Increase the number of washes
conjugate incubation.
with TTBS from two to three
after each antibody incubation
step.
3.
Avidin conjugate concentration
Use avidin conjugate at the
too high.
recommended dilution. Higher
concentrations can lead to higher
background without increasing
sensitivity.
4.
Membrane left in color
Remove membrane from color
development solution too long.
development solution when back-
ground begins to develop and
rinse in distilled water.
5.
Primary or secondary antibody
Perform serial dilutions of
concentration too high.
each antibody to determine
the optimum dilution.
6.
Gelatin blocking solution too old.
Use fresh solution.
11
C.
Color development solution is inactive
1. Reagent improperly stored, or
Store according to
has been exposed to light.
instructions, and check activity
using the activity test procedure
for color development.
D.
HRP Color Development Solution is inactive
1. Light inactivation of color
Make solution fresh each time.
development reagent of H
2
O
2
.
Use stabilized H
2
O
2
.
2. Reagent precipitated out of
Mix solution at room tempera-
solution.
ture. Methanol can react with
Tween-20 to produce a precipi-
tate. This will not interfere with
color development.
E.
Little or no biotinylated standards bound to the membrane
1. Electrophoretic transfer may
Silver stain gel to check that
be incomplete.
all the protein is absent. If pro-
tein is present in the gel, consult
the Trans-Blot cell instruction
manual.
6.3 No Color in Standards, Good Color Development in
Sample
A.
Avidin conjugate is inactive
1. Avidin conjugate has been
Check activity using the activity
improperly stored.
test procedure for conjugate.
Avoid repeated freeze-thaw
cycles by storing the reagent at
4 °C.
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