Bio-Rad Biotinylated Standards - Manual (Page 7)

Section 6
Troubleshooting Guide
6.1 Activity Test for Reagents
A. Activity test for HRP color development solution.
Combine 1.0 ml of the color development solution with 5 µl of concen-
trated avidin-HRP conjugate. The color reaction should develop imme-
diately. If color fails to develop within a few minutes, the color
development solution is inactive. Make up fresh working solution and
repeat the color development assay.
B. Activity test for AP color development solution.
Combine 1.0 ml of the color development solution with 5 µ l full
strength avidin-AP. The color reaction should develop immediately. If
color fails to develop within a few minutes, the color development
solution is inactive. Make up fresh solution and repeat the color devel-
opment assay.
C. Activity test for avidin conjugate solution.
Combine 1.0 ml of the color development solution (tested above) and
1.0 ml of the 1:3,000 dilution of avidin conjugate. A light blue tinge
should develop within 15 minutes. If color fails to develop within 25
minutes, the conjugate solution is suspect. Repeat the procedure with a
freshly prepared dilution of conjugate.
6.2 No Reaction or Weak Color Development
A. Enzyme inactivation
1. Tap water or water deionized Use distilled, deionized water.
by polystyrene resins may Other reagents should be of
inactivate the enzyme. the highest purity.
B. HRP enzyme is inactive
1. Azide is a potent inhibitor of Do not use sodium azide in
HRP enzyme. any of the solutions. If a bacte-
riostat is necessary, use
Thimerosal (merthiolate) at a
0.01% concentration.
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5.3 Protocol for Immune Detection
1. Immerse the membrane at a 45° angle into the proper blocking solu-
tion. Gently agitate the solution, using a shaker platform, for 30 min-
utes to 1 hour.
2. Remove the blocking solution and wash the membrane twice in TTBS,
5 minutes with gentle agitation.
3. Remove wash solution, and add the first antibody solution. Incubate
for 1 hour. Longer incubation times may increase sensitivity, but may
also increase background.
4. Remove the unbound first antibody by washing the membrane twice
for 5 minutes in TTBS with gentle agitation.
5. Remove the wash solution,and add the second antibody, avidin-HRP or
avidin-AP conjugate solution. This solution should contain the appro-
priate dilution of blotting grade second antibody conjugate, protein A
or protein G conjugate. The avidin conjugate is used in a 1:3,000 dilu-
tion. Use approximately 100 ml for full length gels or 50 ml for mini-
gels. Incubate 1 hour with gentle agitation using a shaker platform.
Save 1 ml of the conjugate solution for troubleshooting, if necessary.
6. Remove the conjugate solution. Wash the membrane for 5 minutes in
TTBS with gentle agitation. Repeat.
7. Wash in 100 ml of TBS for 5 minutes. Repeat.
8. Prepare the appropriate color development solution immediately before
use. Immerse the membrane in the development solution. Allow the color
development to proceed until all the bands are detected. If no color devel-
ops, consult the troubleshooting guide. For color development reactions
longer than 45 minutes, cover the incubation tray to prevent fading. Save
1 ml of the color development solution for troubleshooting, if necessary.
9. Stop the development by immersing the membrane in distilled water
for 10 minutes. Change the water at least once.
Note: A small amount of antigen or 1 ng of rabbit, mouse, or human IgG
dotted on one corner of the membrane will develop color if the immune
detection procedure is successful. This is an excellent check on the opera-
tion of the assay and will help you to gauge the rate of color development.
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