Millipore SNAP i.d Manuel utilisateur

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.

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

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