Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 9)

Probable Cause Recommended Solution
4. MPO omitted from BT and 4. When using Zeta-Probe
TTBS solutions for nylon or other nylon membranes,
membranes. prepare BT and TTBS solu-
tions according to the
instructions for nylon mem-
branes only.
Section 4
References
1. Bers, G. and Garfin, D., Biotechniques, 3, 276 (1985).
2. Towbin, H., Staehlin, T and Gordon, J., Proc. Nat. Acad. Sci. USA,
76, 4350 (1979).
3. Glass II, W. F., Briggs, R. C. and Hnilica, L. S., Science, 211, 70
(1981).
4. Glass II, W. F., Briggs, R. C. and Hnilica, L. S., Anal. Biochem., 115,
219 (1981).
5. Karcher, D., Lowenthal, A., Thormar, H. and Noppe, M., J.
Immunol. Methods, 43, 175 (1981).
6. Legocki, R. P. and Verma, D. P. S., Anal. Biochem., 111, 385 (1981).
7. Reiser, J. and Wardale, J., Eur. J. Biochem., 114, 569 (1981).
8. Olmstead, J. B., J. Biol. Chem., 256, 11955 (1981).
9. Symington, J. M., Green, M. and Brackmann, K., Proc. Nat. Acad.
Sci.,USA., 78, 177 (1981).
10. Burnette, W. N., Anal. Biochem., 112, 195 (1981).
11. Hawkes, R., Niday, E. and Gordon, J., Anal. Biochem., 119, 142
(1982).
12. Bayer, E. A. and Wilchek, M., Trends Biochem. Sci,, 3, N257 (1978).
13. Bayer, E. A. and Wilchek, M., Methods Biochem. Anal., 26, 1
(1980).
14. Wilchek, M. and Bayer, E. A., Immunol. Today, 5, 39 (1984).
15. Green, N. M., J. Biochem., 89, 585 (1963).
Methiolate is a registered trademark of Eli Lilly and Co.
Coomassie is a registered trademark of ICI Organics, Inc.
13
Probable Cause Recommended Solution
5. Little or no protein bound to the 5. Consult the Trans-Blot
membrane. instruction manual for proper
electrophoretic transfer proce-
dures. Refer to the Bio-Dot
instruction manual for the prop-
er microfiltration procedure.
a. Amine containing compounds a. Wash membrane thor-
other than membrane bound oughly in BT prior to stain
proteins competing for reagents. ing (see Membrane
Preparation Section 2.1,3).
b. Amine containing compounds b. Wash incubation vessel
or proteins on the surface of the thoroughly before use.
incubation vessel competing Siliconize glass vessels to
for reagents. prevent reagents from bind-
ing to the surface.
c. Proteins bound to membrane c. Increase amount of pro-
may have few accessible tein transferred onto
amine functional groups. membrane. Some pro-
teins may not stain as
intensely as others.
B. High background
1. Insufficient washing after 1. Increase the number of
avidin-HRP incubation. washes with TTBS from
2 to 3 (see Section 2.2,7).
2. Avidin-HRP concentration 2. Use avidin-HRP reagent
too high. recommended 1:1000
dilution. Lower dilution can
lead to background devel-
opment with no increase in
sensitivity.
3. Membrane left in color 3. Remove membrane from
development too long. color development solution
when background begins to
develop and rinse in dis-
tilled water.
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