INSTRUCTIONS
Number Description
21230 Clean-Blot IP Detection Reagent (HRP), 2.5 ml (40 µg/ml)
Storage: Upon receipt store at 4°C. Product is shipped at ambient temperature.
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Note: If using mou IgG 1, perform a dot blot to determine compatibility. Clean-Blot IP Detection
Reagent (HRP) might not detect mou IgG 1.
Introduction
Clean-Blot IP Detection Reagent (HRP) enables Western blot detection of target proteins without interference from denatured IgG. Background signal that can mask the target protein band is often caud by heavy and light chains from IgGs (~50 and 25 kDa). Western blot interference from IgGs results from immunoprecipitation methods that relea antibody with the
antigen and samples types that contain IgGs, such as tissue extracts (e.g., liver, spleen). Secondary antibodies then detect the denatured and blotted IgGs. Clean-Blot IP Detection Reagent (HRP), however, detects only the native antibodies, providing accurate and specific detection of the target antigen.
Procedure for Western Blotting
A. Additional Materials Required • Western blot on nitrocellulo or PVDF membrane
• Blocking buffer such as StartingBlock™ T20 Blocking Buffer (TBS, Product No. 37543 or PBS, Product No. 37539), SuperBlock ® T20 Blocking Buffer (TBS, Product No. 37536 or PBS, Product No. 37516), or 1-5% dry milk • Wash buffer such as Tris-buffered saline (TBS, Product No. 28376) or
phosphate-buffered saline (PBS, Product No. 28372) containing 0.05% Tween ®-20 (Product No. 28320) • Primary antibody specific for antigen of interest • Horradish peroxida (HRP) substrate
•
Film or CCD camera if using a chemiluminescent substrate
B. Antibodies Compatible with Clean-Blot IP Detection Reagent
The Clean-Blot IP Detection Reagent detects the polyclonal and monoclonal antibodies listed in the table below. For best results, test each specific antibody.
Species Monoclonal Isotype(s) Bovine IgG 2 Goat IgG 2
Human IgG 1, IgG 2, IgG 4 Mou IgG 2a, IgG 2b, IgG 3 Rat IgG 2c离婚协议样本
Rabbit Total IgG Sheep IgG 2
Clean-Blot™ IP Detection Reagent
(HRP)
C. Concentration Guidelines for the Clean-Blot IP Detection Reagent
Note: The dilution ranges are guidelines. For best results, optimize the dilution for each specific experiment. HRP Substrate
Clean-Blot IP Detection Reagent Dilution Range SuperSignal ® West Femto Chemiluminescent Substrate 1:200 to 1:4,000
(e.g., for 1:4,000 dilution, add 2.5 µl of detection reagent to 10 ml of blocking buffer) SuperSignal West Dura
Chemiluminescent Substrate 1:200 to 1:2,000
(e.g., for 1:2,000 dilution, add 5 µl of detection reagent to 10 ml of blocking buffer) SuperSignal West Pico
Chemiluminescent Substrate 1:40 to 1:1,000
(e.g., for 1:1,000 dilution, add 10 µl of detection reagent to 10 ml of blocking buffer) Pierce ® ECL Western Blotting Substrate 1:40 to 1:400
(e.g., for 1:400 dilution, add 25 µl of detection reagent to 10 ml of blocking buffer)
D. Western Blot Detection
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1. Dilute primary antibody to appropriate concentration in blocking buffer.
Note: If using mou IgG 1, perform a dot blot to determine compatibility. Clean-Blot IP Detection Reagent (HRP) might not detect mou IgG 1.
2. Add the diluted primary antibody to the blot and incubate for 1 hour at room temperature or overnight at 4°C.
3. Wash blot with Wash Buffer.
4. Dilute the Clean-Blot IP Detection Reagent (HRP) with blocking buffer.
Note: See table above for dilution ranges. For best results, optimize the dilution for each specific experiment. 5. Add the diluted detection reagent to the blot and incubate for 1 hour at room temperature or overnight at 4°C 6. Wash blot with wash buffer.
7. Prepare and add the HRP substrate according to the manufacturer’s instructions. 8. Expo to blot to film or CCD camera.海螵蛸
Troubleshooting
Problem Possible Cau Solution Too much HRP in the system Dilute the Clean-Blot IP Detection Reagent Too much primary antibody Dilute primary antibody
Nonspecific bands SDS caud nonspecific binding to
protein bands
Do not u SDS during immunoassay procedure Speckled background on film Aggregate formation in the Clean-Blot IP Detection Reagent (HRP) Filter detection reagent through a 0.2 µm filter or centrifuge and u supernatant Too much or not enough HRP in the
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system
Optimize the Clean-Blot IP Detection Reagent Insufficient quantities of antigen or
antibody
Increa concentration of antibody or antigen Weak or no signal Inefficient protein transfer Optimize transfer
Ud too much detection reagent Dilute the Clean-Blot IP Detection Reagent
Inadequate blocking Optimize blocking conditions Inappropriate blocking reagent Try a different blocking reagent Inadequate washing Increa length, number or volume of washes Film was overexpod Decrea exposure time or u Pierce Background
Eliminator (Product No. 21065)男女合唱的歌曲有哪些
High background Antigen or antibody is too concentrated Dilute the antigen or antibody
Problem Possible Cau Solution
Increa reducing reagent concentration Boil sample to aid in reduction of IgG disulfide bonds
Detection of denatured and blotted IgG All IgG in the sample was not
denatured U denaturing electrophoresis conditions Sample does not contain antigen or
does not contain a detectable quantity
of antigen
Optimize expression and lysis procedures Antibody does not recognize antigen U a different primary antibody Antigen of interest was not detected Primary antibody is not compatible Verify target by performing a dot blot using a
traditional condary antibody Additional Information龟缩
Plea visit the website for additional information on this product including the following: • Tech Tip protocol: Optimization of antigen and antibody concentrations for Western blots • Tech Tip protocol: Optimization of blocking buffer and cross-reactivity determination
Related Products
21233 Clean-Blot IP Detection Reagent (AP), 2.5 ml 34090 CL-XPosure™ Film, 5” × 7” sheets, 100 sheets/pkg
32106 Pierce ECL Western Blotting Substrate , 500 ml
34080 SuperSignal West Pico Chemiluminescent Substrate, 500 ml 34075 SuperSignal West Dura Extended Duration Substrate, 100 ml 34095 SuperSignal West Femto Maximum Sensitivity Substrate, 100 ml 21059 Restore™ Western Blot Stripping Buffer, 500 ml
21065 Pierce Background Eliminator Kit, for eliminating background from X-ray film 37515 SuperBlock (PBS) Blocking Buffer, 1 L 37535 SuperBlock (TBS) Blocking Buffer, 1 L 37542 StartingBlock (TBS) Blocking Buffer , 1 L 37538 StartingBlock (PBS) Blocking Buffer , 1 L
88018 Nitrocellulo Membrane, 0.45 µm, 33 cm × 3 m, 1 roll 77010 Nitrocellulo Membrane, 0.45 µm, 8 × 12 cm, 25/pkg. 88025 Nitrocellulo Membrane, 0.45 µm, 8 × 8 cm, 15/pkg. 88600 Western Blotting Filter Paper, 8 cm × 10.5 cm, 100 sheets
24580 MemCode™ Reversible Protein Stain Kit for Nitrocellulo Membranes 24585 MemCode Reversible Protein Stain Kit for PVDF Membranes
28320 Surfact-Amps ® 20, 6 × 10 ml ampules containing 10% solutions of Tween-20 Detergent 28374 BupH™ Modified Dulbecco’s PBS Packs , 40 packs, each yielding 500 ml 28376 BupH Tris Buffered Saline Packs , 40 packs, each yielding 500 ml
SuperSignal®Technology is protected by U.S. Patent # 6,432,662
Tween®is a registered trademark of ICI Americas.
This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as t forth in the Product documentation, specifications and/or accompanying package inrts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwi expressly authorized in writing, Products are supplied for rearch u only. No claim of suitability for u in applications regulated by FDA is made. The warranty provided herein is valid only when ud by properly trained individuals. Unless otherwi stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchar of the Product (“Buyer”).
No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpo, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s).
There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misu, fault or negligence of or by Buyer, (iii) u of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products.
Current versions of product instructions are available at /pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.
© 2008 Thermo Fisher Scientific Inc. All rights rerved. Unless otherwi indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its
subsidiaries. Printed in the USA.
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