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Summary of Contents for Millipore Sigma SNAP i.d. 2.0
- Page 1 User Guide ® SNAP i.d. Protein Detection System for Western Blotting The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the US and Canada. www.sigmaaldrich.com...
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Page 2: Table Of Contents
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. -
Page 3: Introduction
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. -
Page 4: Parts And Functions Of The Snap I.d
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 ® Base Unit (1) Accepts frames and facilitates immunodetection Tubing Assembly Kit (1) Connects system to vacuum source Blot roller (1) -
Page 5: Symbols Used In This User Guide
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 Symbol Definition Warning alerts you to actions that Serial number may cause personal injury or pose a physical threat. -
Page 6: General Guidelines
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. •... -
Page 7: Optimization Guidelines
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. - Page 8 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. Standard SNAP i.d. ®...
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Page 9: Blot Blocking
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). •... -
Page 10: How To Use The Snap I.d
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. -
Page 11: Blot Assembly And General Protocol
Blot Assembly and General Protocol 1. Hold the blot holder by the support layer (blue edges) and wet the membrane layer (white) with distilled water in the wetting tray provided. Do not wet the support layer. Place the wetted blot holder on the rolling pad. 2. - Page 12 6. Apply appropriate volume of primary antibody across the surface of the blot holder (2.5 mL for MultiBlot, 5 mL for Mini blot, or 10 mL for Midi blot). 7. Incubate for 10 minutes at room temperature. Solution will be absorbed into the blot holder and surface may appear dry.
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Page 13: Extended Primary Antibody Incubation: One Hour To Overnight
Extended Primary Antibody Incubation: One Hour to Overnight 1. Perform steps 1 through 5 of the General Protocol. 2. Add 2.5 mL (for MultiBlot), 5 mL (for Mini blot), or 10 mL (for Midi blot) of 1X primary antibody (concentration normally used during standard immunodetection). -
Page 14: Proof Of Performance
Proof of Performance Figure 1. Immunodetection of β-Tubulin: SNAP i.d. 2.0 System vs. SNAP i.d. System (first generation) vs. Standard Immunodetection ® ® a. SNAP i.d. 2.0 Protein Detection b. SNAP i.d. Protein Detection c. Standard ® ® System Midi Blot System First generation, single well Immunodetection 3T3 Control 3T3 Anisomycin 3T3 PDGF... - Page 15 Figure 3. Immunodetection of Tau-1 protein: SNAP i.d. 2.0 System (MultiBlot, Midi, and Mini) vs. Standard Immunodetection ® b. SNAP i.d. c. SNAP i.d. d. Standard ® ® a. SNAP i.d. 2.0 MultiBlot Mini Blot Midi Blot Immunodetection ® Alzheimer’s brain Healthy brain Alzheimer’s brain Healthy brain Alzheimer’s brain...
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Page 16: Maintaining The Snap I.d. ® 2.0 System
Figure 5. Extended Incubation (1 Hour): SNAP i.d. 2.0 System 1 Hour Protocol vs. SNAP i.d. 2.0 System Standard Protocol vs. ® ® Standard Immunodetection a. SNAP i.d. 2.0 Protein Detection ® b. SNAP i.d. 2.0 Protein Detection c. Standard ®... -
Page 17: Troubleshooting
Troubleshooting Symptom Cause Corrective Action Vacuum control knobs stick Inadequate cleaning Flush system with distilled water. Blot holders do not empty Inadequate vacuum Make sure tubing connection between system and vacuum or empty slowly when source is secure. vacuum is applied MultiBlot frame was used to When running a single blot in the MultiBlot frame, place well process a single blot, and well... -
Page 18: Specifications
Specifications Dimensions Base (length × width × height) 40.6 cm (16 in.) × 32.4 cm (12.75 in.) × 8.9 cm (3.5 in.) Weight (approximate) 1.5 kg (3.3 lb) MultiBlot frame with lid (length × width × height) 19.7 cm (7.75 in.) × 14.7 cm (5.8 in.) × 3.6 cm (1.4 in.) MultiBlot holder 11.4 cm (4.5 in.) ×... -
Page 19: Ordering Information
Ordering Information Purchase products online at www.sigmaaldrich.com/products. Product Description Cat. No. Qty/Pk Base System SNAP i.d. 2.0 Base SNAP2BASE ® Base unit (1) Tubing assembly kit (1) Blot roller (1) Rolling pad (1) Wetting trays (2) Antibody collection trays (2) Quick-Start Guide (1) Components for Western Blotting Procedures SNAP i.d. - Page 20 Product Description Cat. No. Qty/Pk Reagents for Western Blotting Immobilon Forte Western HRP Substrate WBLUF0500 500 mL ® Immobilon Crescendo Western HRP Substrate WBLUR0500 500 mL ® Immobilon Classico Western HRP Substrate WBLUC0500 500 mL ® Immobilon Western HRP Substrate WBKLS0500 500 mL ®...
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Page 21: Notice
Notice We provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose.
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