Antibody Volume and Concentration
Most users will be able to use the same amount of antibody, but in 1/3 to 1/5 the
■
volume at 3
–
5 fold higher concentration.
Standard
Immunodetection
SNAP i.d.
Immunodetection
Mass of antibody
required
1 µg
1 µg
Stock solution
1 mg/mL
1 mg/mL
Diluted stock
1:10,000 (0.1 µg/mL)
1:3,333 (0.33 µg/mL)
Volume required for assay
10 mL
3 mL
Antibody used
0.1 µg/mL × 10 mL = 1 µg
0.33 µg/mL × 3 mL = 1 µg
This guideline is intended as a starting point to develop the final antibody concentration necessary for desired
performance. Because each antibody is different, it may be necessary to adjust the blot exposure time, antigen
load or both.
Blot Blocking Concentration
The use of non-fat/low fat dry milk at concentrations higher than 0.5% is not recom-
■
mended, as this will result in clogging of the blot holder and prevent reagent flow.
Blocking agents should be prepared in tris or phosphate buffered saline solutions contain-
■
ing 0.1% Tween
20 surfactant, to reduce surface tension and ensure even distribution of
blocking agent across the blot holder surface.
The SNAP i.d. system is compatible with the most commonly used blocking agents. Refer
■
to User Guide for complete list with recommended concentrations.
In order to insure optimal flow through the blot holder, it is essential that blocking solu
-
■
tions be completely solubilized and free of all particulate matter. In some cases, it may be
necessary to reduce the concentration of the blocking agent to achieve the required flow.
Blocking, Antibody and Wash Recommended Volumes
Single well
Double well
Triple well
Blocking solution volume
30 mL/well
15 mL/well
10 mL/well
Antibody volume
3 mL/well
1.5 mL/well
1 mL/well
Wash buffer* volume
30 mL/well
15 mL/well
10 mL/well
It is not necessary to use all the wells of double and triple well blot holders, but unused wells must
be wet out with Milli-Q water.
* Tris or phosphate buffered saline solutions, supplemented with 0.1% Tween
®
20 surfactant.
OPTIMIZATION GUIDELINES
AVID QS 021108.indd 2
2/11/2008 6:40:46 PM
