Millipore Sigma SNAP i.d. 2.0 Manuale utente

The life science business of

Merck KGaA, Darmstadt, Germany

operates as MilliporeSigma in

the US and Canada.

www.sigmaaldrich.com

User Guide

SNAP i.d.® 2.0

Protein Detection System

for Western Blotting

CONTENTS

Introduction .........................................................................................................................1

SNAP i.d.®2.0 Protein Detection System Features ....................................................................................1

Parts and Functions of the SNAP i.d.®2.0 System .................................................................2

Symbols Used in this User Guide...........................................................................................3

Materials Required but Not Supplied.....................................................................................3

General Guidelines ...............................................................................................................4

Optimization Guidelines........................................................................................................5

Antibodies ..........................................................................................................................................5

Blot Blocking ........................................................................................................................7

How to Use the SNAP i.d.®2.0 System ..................................................................................8

System Setup......................................................................................................................................8

Blot Assembly and General Protocol........................................................................................................9

Extended Primary Antibody Incubation: One Hour to Overnight .........................................11

Antibody Recovery..............................................................................................................11

Proof of Performance..........................................................................................................12

Maintaining the SNAP i.d.®2.0 System................................................................................14

Cleaning Protocol ...............................................................................................................................14

Component Re-use.............................................................................................................................14

Troubleshooting .................................................................................................................15

Specifications .....................................................................................................................16

Chemical Compatibility .......................................................................................................16

Storage ..............................................................................................................................16

Ordering Information .........................................................................................................17

Notice.................................................................................................................................19

Contact Information ...........................................................................................................19

Technical Assistance...........................................................................................................19

Standard Warranty .............................................................................................................19

Conformance to Pressure Equipment Directive ...................................................................19

00000825W REV 05/19 1 of 19

Introduction

The new and improved SNAP i.d.®2.0 Protein Detection System is the second generation of the SNAP i.d.®method

for detecting immunoreactive proteins on Western blots. With this unique vacuum-driven system, the length of

time required for immunodetection is greatly reduced. What previously took 4 to 24 hours with traditional Western

blotting methods now takes only 30 minutes with no loss of signal intensity or reduction in blot quality. All

immunodetection steps after protein transfer to a membrane (i.e., blocking, washing, and primary and secondary

antibody incubations) can be performed with the SNAP i.d.®2.0 system.

Sample Prep

0.5–2

hours

0.5–1

hours

1–2.5

hours

1 hour 3 hours 15 minutes

Electro-

phoresis

Membrane

Transfer Blocking

Antibody

Incubation Detection

SNAP i.d.®2.0 System

30 minutes

SNAP i.d.®2.0 Protein Detection System Features

• Processing of up to four blots at a time on a vacuum base with two individually controlled sides

• Three removable blot holding frame sizes: MultiBlot, Mini, and Midi, to accommodate different blot sizes

• Disposable blot holders, sized for MultiBlot, Mini, and Midi frames

• Blot spacer required with first generation SNAP i.d.®system is now integrated into the new blot holder

• Extended and off-line blot processing options: frames have lids and can be removed from the base for

extended incubation (one hour to overnight), incubation in a shaker, or incubation at 4 °C

• Stackable frames so multiple blots can be processed at the same time

• 30-minute immunodetection with uniform signal across the blot

• Greater than 80% antibody recovery using the SNAP i.d.®2.0 Antibody Collection Trays

• Compatible with nitrocellulose and polyvinylidene fluoride (PVDF) membranes

• Works with the most blocking buffers and visualization methodologies (e.g. chemiluminescence, fluorescence,

or colorimetric)

The SNAP i.d.®2.0 Protein Detection System is intended for research use only. It is not for use in

diagnostic procedures.

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Parts and Functions of the SNAP i.d.®2.0 System

The SNAP i.d.®2.0 system consists of the following components:

Part Function Cat. No.

SNAP i.d.®2.0 Base Kit (includes the following components) SNAP2BASE

ABase Unit (1) Accepts frames and facilitates immunodetection

BTubing Assembly Kit (1) Connects system to vacuum source

CBlot roller (1) Eliminates air bubbles between the blot and blot holder

DRolling pad (1) Provides smooth surface for assembling blot

EWetting tray (2) Used for wetting out blot holder and blot

FAntibody Collection Tray (2) Collects antibody for recycling

GQuick Start Guide (not shown)

HSNAP i.d.®2.0 MultiBlot Holding

Frame and Lid

Accepts MultiBlot holders for blot incubation SNAP2FRMB01

ISNAP i.d.®2.0 Mini Blot Holding

Frame and Lid

Accepts Mini blot holders for blot incubation SNAP2FRMN01

SNAP2FRMN02

JSNAP i.d.®2.0 Midi Blot Holding

Frame and Lid

Accepts Midi blot holders for blot incubation SNAP2FRMD01

SNAP2FRMD02

KSNAP i.d.®2.0 MultiBlot Holder

(includes 2 MultiBlot well blanks)

Accepts up to 4.5 × 8.4 cm blots

Blank is used to block empty chamber when running only

one blot

SNAP2BHMB050

LSNAP i.d.®2.0 Mini Blot Holder Accepts up to 7.5 × 8.4 cm blots SNAP2BHMN0100

MSNAP i.d.®2.0 Midi Blot Holder Accepts up to 8.5 × 13.5 cm blots SNAP2BHMD0100

00000825W REV 05/19 3 of 19

Symbols Used in this User Guide

The following symbols are used throughout this user guide and/or on product labels, and the user shall abide by

indicated requirements:

Symbol Definition

YWarning alerts you to actions that

may cause personal injury or pose a

physical threat.

iRead the documentation

hCatalogue number

Symbol Definition

fSerial number

gLot number

MManufacturer

DDo not re-use

Materials Required but Not Supplied

• Vacuum source: Pump or other uniform vacuum source that can deliver a sustained minimum pressure of

410 millibar (12 inHg) and 34 L/min. Refer to Ordering Information for pump catalogue numbers.

NOTE: Our WP61 series Chemical Duty Pumps can be used, but may require longer processing times.

• One liter or larger vacuum flask with stopper (for waste collection). A Millex®-FA50 filter (or equivalent)

is recommended between the vacuum flask and the vacuum source to protect the vacuum source from

contamination.

• Vacuum tubing to connect vacuum flask to vacuum source

• Forceps

• Blocking reagent such as non-fat/low fat dry milk (0.5% or less), casein, bovine serum albumin (BSA) or other

commercially available blocking agents such as bløk®-CH buffer (see Table 5)

• Antibodies (monoclonal and/or polyclonal)

• Detection reagents

• Wash buffer: tris- or phosphate-buffered saline solution, pH 7.4, supplemented with Tween®20 surfactant (TBST

or PBST)

• Blot with transferred proteins

Blot Holder Size Maximum Blot Size (cm)

MultiBlot 4.5 × 8.4

Mini 7.5 × 8.4

Midi 8.5 × 13.5

00000825W REV 05/19 4 of 19

General Guidelines

• If proteins have been transferred to PVDF membrane that has been dried out, re-wet the blot with 100%

methanol, then rinse with distilled water prior assembly. If proteins have been transferred to nitrocellulose

membrane that has been dried out, re-wet the blot with distilled water.

• For most applications and for most antibodies, a 0.5% solution of non-fat/low fat dry milk provides sufficient

blocking of the membrane.

NOTE: Do not use dry milk concentrations higher than 0.5% because it can cause clogging of the blot holder

membrane. If using other blocking solutions, refer to Table 5 for compatibility and recommended concentration.

• Always use 0.1% Tween®20 surfactant in the washing, blocking, and antibody buffers. This will reduce the

surface tension of solutions and ensure even distribution of the antibody in the blot holder during incubation.

• Since immunodetection in the SNAP i.d.®2.0 system is performed in a short period of time, it is recommended

that primary and secondary antibody dilutions be prepared prior to starting the procedure.

• The total volume of diluted antibody required is 2.5 mL for the MultiBlot holder, 5 mL for the Mini blot holder,

and 10 mL for the Midi blot holder.

• Four washes of 30 mL each (15 mL for MultiBlot) are needed to ensure the complete wash of the blot after each

incubation step. Refer to Table 2 for details.

• Stripping blots in the SNAP i.d.

2.0 system is not recommended. Blots that have been stripped outside of the

system following a standard protocol can be reprobed in the SNAP i.d.

2.0 system starting with the blocking step.

• If stripping is not required (for example, if detecting with a different secondary antibody), blots can be reprobed

immediately in the SNAP i.d.®2.0 system after a quick wash.

00000825W REV 05/19 5 of 19

Optimization Guidelines

Antibodies

Optimization guidelines have been developed to enable users to convert the antibody concentrations used in

standard immunodetection assays into concentrations applicable to the SNAP i.d.®2.0 system. Most users will

be able to use the same amount of antibody, but in less volume at a higher concentration than for standard

immunodetection. However, when using the extended antibody protocol (page 12), it is possible to use the same

concentration, volume, and time for the primary antibody that is regularly used for standard immunodetection,

followed by the secondary antibody at a higher concentration for a shorter period of time.

Table 1. How to calculate the antibody concentration required for the SNAP i.d.®2.0 system based on the

concentration used for standard immunodetection

Standard

Immunodetection

SNAP i.d.®2.0 Immunodetection

MultiBlot Mini Blot Midi Blot

Ab stock concentration 1 mg/mL 1 mg/mL 1 mg/mL 1 mg/mL

Mass of antibody required 1 µg 0.25 µg 0.5 µg 1 µg

Volume of Ab used 30 mL 2.5 mL 5 mL 10 mL

Final Ab dilution 1 : 30,000 1 : 10,000 1 : 10,000 1 : 10,000

Antibody stock used 1 µL 0.25 µL 0.5 µL 1 µL

Because each antibody is unique, and the sensitivity of detection reagents varies, it may be necessary to adjust

the antibody or antigen concentration, the type or sensitivity of the detection reagent used, or the blot X-ray

exposure time. Please note that this guideline is intended as a starting point to develop the final antibody

concentration for the desired performance.

Table 2. Blocking, Antibody, and Wash Recommended Volumes

SNAP i.d.®2.0

MultiBlot Frame

SNAP i.d.®2.0

Mini Frame

SNAP i.d.®2.0

Midi Frame

Blocking solution volume 15 mL per well 30 mL 30 mL

Antibody volume 2.5 mL per well 5 mL 10 mL

Wash buffer* volume 4 × 15 mL each per well 4 × 30 mL each 4 × 30 mL each

* Tris- or phosphate-buffered saline solutions, supplemented with 0.1% Tween®20 surfactant

When calculating the amount of antibody required for immunodetection, three components are critical for

successful detection of the protein and a good quality blot (low background, high signal):

• Type of sample and concentration loaded on the gel

• Primary and secondary antibody concentration

• Type and sensitivity of the detection reagent

All of the antibodies in the following list were tested using Luminata™ Forte chemiluminescence detection reagent.

For the SNAP i.d.®2.0 system, the antibody concentration is higher than in standard immunodetection, but in less

volume.

00000825W REV 05/19 6 of 19

Table 3. Examples of Primary Antibody Dilution in Standard Immunodetection vs.

SNAP i.d.®2.0 Immunodetection

NOTE: All antibodies were tested using 0.5% NFDM as the blocking reagent and Luminata™ Forte Western HRP

Substrate as the chemiluminescent reagent.

Primary AntibodyaHostbCat. No. Protein MW

Standard

Ab dilution

SNAP i.d.®2.0

Ab dilution

Anti-Akt1/PKBα Rb 05-796 60 1 : 1,200–1 : 2,000 1 : 400

Anti-CREB Rb 06-863 43 1 : 600–1 : 1,000 1 : 200

Anti-Caspase-3 Rb AB3623 17 1 : 1,200–1 : 2,000 1 : 400

Anti-Cyclin D1 Rb 04-1151 36 1 : 600–1 : 1,000 1 : 200

Anti-EGFR M 05-104 170 1 : 600–1 : 1,000 1 : 200

Anti-MAP Kinase 1/2 ErK1/2 Rb 06-182 44-42 1 : 1,500–1 : 2,500 1 : 500

Anti-erbB2 M 04-291 185 1 : 600–1 : 1,000 1 : 200

Anti-GAPDH M MAB374 38 1 : 30,000–1 : 50,000 1 : 10,000

Anti-GST M1 (mu) Rb ABN19 26 1 : 600–1 : 1,000 1 : 200

Anti-GFAP M MAB3402 50 1 : 1,200–1 : 2,000 1 : 400

Anti-Glutamate receptor 1 Rb AB1504 100 1 : 1,200–1 : 2,000 1 : 400

Anti-Huntington protein M MAB2166 350-400 1 : 1,200–1 : 2,000 1 : 400

Anti-Integrin α5 Rb AB1928 114 1 : 3,000–1 : 5,000 1 : 1,000

Anti-MGluR5 Rb AB5675 132 1 : 600–1 : 1,000 1 : 200

Anti-NFkB p52 Rb 06-413 100 & 52 1 : 1,200–1 : 2,000 1 : 400

Anti-NMDAR1 Rb AB9864 120 1 : 1,200–1 : 2,000 1 : 400

Anti-P53 (N-term) Rb 04-1083 53 1 : 600–1 : 1,000 1 : 200

Anti-pan-Cadherin Rb ABT35 120-130 1 : 1,200–1 : 2,000 1 : 400

Anti-PP2A M 05-421 36 1 : 3,000–1 : 5,000 1 : 1,000

Anti-PTEN M 04-035 55 1 : 600–1 : 1,000 1 : 200

Anti-Pyk2 Rb 06-559 114 1 : 600–1 : 1,000 1 : 200

Anti-Ras M 05-516 21 1 : 1,500–1 : 2,500 1 : 500

Anti-Rac1 M 05-389 21 1 : 600–1 : 1,000 1 : 200

Anti-STAT1 M 05-987 91 1 : 300–1 : 500 1 : 100

Anti-Tau1 M MAB3420 52-68 1 : 1,200–1 : 2,000 1 : 400

Anti-β-Tubulin M MAB3408 50 1 : 3,000–1 : 5,000 1 : 1,000

aGo to www.sigmaaldrich.com/antibodies for details

bM = Mouse, Rb = Rabbit

Table 4. Examples of Secondary Antibody Dilution in Standard Immunodetection

vs. SNAP i.d.®2.0 Immunodetection

Secondary AntibodyaHostbCat. No.

Standard

Ab dilution

SNAP i.d.®2.0

Ab dilution

Anti-Mouse IgG Gt AP124P 1 : 50,000 1 : 10,000

Anti-Rabbit IgG Gt AP132P 1 : 200,000 1 : 30,000

Anti-Sheep IgG Rb AP147P 1 : 50,000 1 : 20,000

Anti-Goat IgG Rb AP106P 1 : 80,000 1 : 10,000

a Horseradish peroxidase conjugate

b Gt = Goat, Rb = Rabbit

00000825W REV 05/19 7 of 19

Blot Blocking

• The SNAP i.d.®2.0 system is compatible with most commonly used blocking agents including non-fat/low fat dry

milk, bovine serum albumin (BSA) and casein. Many other commercially available blockers are also compatible

(refer to Table 5).

• In order to ensure optimal flow through the blot holder, it is essential that blocking solutions 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.

• The use of non-fat/low fat dry milk at concentrations higher than 0.5% is not recommended.

• Blocking agents, should be prepared in tris- or phosphate-buffered saline solution containing 0.1% Tween®20

surfactant, to reduce surface tension and ensure even distribution of blocking agent across the blot holder

surface.

• To ensure even distribution of the antibody in the incubation step, dilute the antibody in blocking solution that

contains Tween®20 surfactant.

Table 5. Compatibility with Blocking Reagents

Blocker Compatible

Recommended

Concentration

Non-fat/low fat dry milk (NFDM) Yes, ≤ 0.5% 0.5%

Immunoblot Blocking Reagent, NFDM (cat. no. 20-200) Yes, ≤ 0.5% 0.5%

BLOT-QuickBlocker™ Reagent (cat. no. WB57-175GM) Yes 0.5%

bløk®-PO Buffer (cat. no. WBAVDP001) Yes Undiluted

bløk®-FL Buffer (cat. no. WBAVDFL01) Yes Undiluted

bløk®-CH Buffer (cat. no. WBAVDCH01) Yes Undiluted

N-Z-Amine®AS (Fluka®)Yes, ≤ 5% 1%

Probumin®Bovine Serum Albumin (BSA) (cat. no. 820473) Yes, ≤ 5% 1%

5% Alkali-soluble Casein (Novagen®cat. no. 70955) Yes 1%

SEA BLOCK Blocking Buffer (Pierce®) Yes Undiluted

SuperBlock®Blocking Buffer with Tween®20 surfactant (Pierce®) Yes Undiluted

LI-COR®Odyssey®Blocking Buffer Yes Undiluted

PVP-40 (Polyvinylpyrrolidone) Yes, ≤ 1% 1%

Gelatin No N/A

Immobilon®Signal Enhancer (cat. no. WBSH0500) Yes Undiluted

00000825W REV 05/19 8 of 19

How to Use the SNAP i.d.®2.0 System

System Setup

1. Place the SNAP i.d.®2.0 base on a level bench top.

2. Attach the vacuum tubing to the back of the system by pushing the coupling insert on the end of the tubing

into the quick disconnect fitting at the back of the system base until it clicks.

Push tab up to

insert or release

coupling To vacuum

NOTE: To disconnect the tubing, push the tab below the tubing connector up with the index finger and pull

tubing out.

3. Connect the other end of the tubing to a vacuum source. Use a one-liter vacuum flask as a trap and a

Millex®-FA50 filter (cat. no. SLFA05010) to protect the vacuum source from contamination, as shown below.

To SNAP i.d.®

2.0 System

Tra p Vacuum source

Vacuum

protection filter

NOTE: Any vacuum source that can deliver 410 millibar (12 inHg) and 34 L/min is sufficient. If the vacuum

source operates at higher than 410 millibar, the SNAP i.d.®2.0 system will automatically regulate the vacuum

pressure.

If the vacuum source is insufficient, the flow rate through the system may be inconsistent, resulting in longer

processing times and/or poor antibody recovery.

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