Millipore SNAP i.d User manual

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.
Apply appropriate volume of secondary antibody (see12. OPTIMIZATION
GUIDELINES on reverse side) evenly across the blot holder surface.
Incubate for 10 minutes at room temperature. Again, solution will be ab-
sorbed into the blot holder and surface may appear dry.Apply vacuum.
IMPORTANT: Do not apply vacuum until after the 10-minute
incubation.
With vacuum on, wash 3 times with buffer. See13. OPTIMIZATION
GUIDELINES on reverse side for volumes.
TURN VACUUM OFF.
Open lid of system and place8. blot holder in chamber, aligning
blot holder tabs with notches of
chamber. Close and latch lid.
Add volume of blocking solution9. 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 GUIDE-
LINES 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 ap-
pear dry.Apply vacuum.
IMPORTANT: Do not ap-
ply vacuum until after the
10-minute incubation.
++
With vacuum running11. continuously, wash 3 times
with wash buffer. See
OPTIMIZATION GUIDE-
LINES on reverse side for
volumes.
TURN VACUUM OFF.
3X
Remove blot and incubate with the14. appropriate detection reagent such
as Immobilon®HRP, or, if using Milli-
pore uorescently labeled antibod-
ies, visualize.
Squeeze rmly at base of tab 7. area to secure lid.
Close the blot holder lid.6.
Thoroughly wet the white2. 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 spacer (wetting not5. necessary) on top of the blot
membrane and roll again to
ensure contact of spacer with
blot membrane.
Roll blot membrane gently to4. remove air bubbles.
Open the blot holder lid, taking1. care not to damage the inner
white surface.
OVERVIEW OF PROCEDURE
Before using the SNAP i.d. Protein Detection System, please read the User Guide completely.
Place the pre-wet blot in the3. center of the blot holder with
the protein side down. The blot
membrane should not exceed
size specied in the User Guide.
00103870 Rev. A, 01/08
AVID QS 021108.indd 1 2/11/2008 6:40:46 PM

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 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.
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.
Blocking agents should be prepared in tris or phosphate buffered saline solutions contain-■ing 0.1% Tween20 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.
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