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OVERVIEW OF PROCEDURE
Before using the SNAP i.d. Protein Detection System, please read the User Guide completely.
Open the blot holder lid, taking 1.
care not to damage the inner
white surface.
Thoroughly wet the white 2.
surface of the blot holder with
Milli-Q® water.
NOTE: If using only one
well of a double or triple
well blot holder, the unused
well(s) must also be wet.
Place the pre-wet blot in the 3.
center of the blot holder with
the protein side down. The blot
membrane should not exceed
size specied in the User Guide.
Roll blot membrane gently to 4.
remove air bubbles.
Open lid of system and place 8.
blot holder in chamber, aligning
blot holder tabs with notches of
chamber. Close and latch lid.
Add volume of blocking solution 9.
as indicated under OPTIMIZA-
TION GUIDELINES on reverse
side. Using knobs on the system,
apply vacuum until well(s) are
completely empty.
TURN VACUUM OFF.
Add volume of primary anti-10.
body as indicated under
OPTIMIZATION GUIDELINES on reverse side.
Antibody solution must evenly
cover entire blot holder
surface.
Incubate for 10 minutes at
room temperature. Solution
will be absorbed into the blot
holder and surface may appear dry. Apply vacuum.
IMPORTANT: Do not apply vacuum until after the
10-minute incubation.
Place the spacer (wetting not 5.
necessary) on top of the blot
membrane and roll again to
ensure contact of spacer with
blot membrane.
Close the blot holder lid.6.
Squeeze rmly at base of tab 7.
area to secure lid.
00103870 Rev. A, 01/08
With vacuum running 11.
continuously, wash 3 times
with wash buffer. See
OPTIMIZATION GUIDELINES on reverse side for
volumes.
TURN VACUUM OFF.
Apply appropriate volume of secondary antibody (see 12. OPTIMIZATION
GUIDELINES on reverse side) evenly across the blot holder surface.
Incubate for 10 minutes at room temperature. Again, solution will be absorbed into the blot holder and surface may appear dry. Apply vacuum.
IMPORTANT: Do not apply vacuum until after the 10-minute
With vacuum on, wash 3 times with buffer. See 13. OPTIMIZATION
GUIDELINES on reverse side for volumes.
TURN VACUUM OFF.
Remove blot and incubate with the 14.
appropriate detection reagent such
as Immobilon® HRP, or, if using Millipore uorescently labeled antibodies, visualize.
Millipore, Milli-Q and Immobilon are registered trademarks of
Millipore Corporation.
SNAP i.d. and the M mark are trademarks of Millipore Corporation.
Tween is a registered trademark of ICI Americas Inc.
incubation.
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OPTIMIZATION GUIDELINES
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
Wash buffer* volume 30 mL/well 15 mL/well 10 mL/well
* Tris or phosphate buffered saline solutions, supplemented with 0.1% Tween
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.
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 ow.
3 mL/well 1.5 mL/well 1 mL/well
®
20 surfactant.
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 ow 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 ow.
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
Mass of antibody
required
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 nal 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.
1 µg 1 µg
SNAP i.d.
Immunodetection
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