ab Streptavidin Conjugation Kit Protocol
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1 ab Streptavidin Conjugation Kit Protocol Instructions for Use Antibody and protein modification This product is for research use only and is not intended for diagnostic use. Version 6 Date modified 24 November 2016
2 Table of Contents INTRODUCTION 1. BACKGROUND 1 2. ASSAY SUMMARY 2 GENERAL INFORMATION 3. PRECAUTIONS 3 4. STORAGE AND STABILITY 3 5. LIMITATIONS 3 6. MATERIALS SUPPLIED 4 7. MATERIALS REQUIERED NOT SUPPLIED 5 8. TECHNICAL HINTS 5 PREPARATION 9. REAGENT PREPARATION SAMPLE PREPARATION 6 PROCEDURE 11. CONJUGATION PROCEDURE 7 RESOURCES 12. NOTES 8
3 INTRODUCTION 1. BACKGROUND Abcam s Streptavidin Conjugation Kit provides a simple and quick process to conjugate your primary antibodies with Streptavidin. The kit enables the direct labeling of antibodies, proteins, peptides or other biomolecules with an available amine group for use in R&D applications, drug discovery and the development of diagnostic kits. Simply pipette the antibody or other biomolecule into the vial of Streptavidin Conjugation Mix and incubate for 3 hours. The antibody to be labelled should be purified, in an appropriate buffer for conjugation and at a suitable concentration, as described in section 10. If not, consider using our antibody purification and concentration kits. The conjugated antibody can be used straight away in WB, ELISA, IHC etc The Kit is available in 3 sizes 30µg (3 x 10 µg) sufficient for labeling up to 30µg of antibody 300µg (3 x 100µg) sufficient for labeling up to 300µg of antibody 1mg (1 x 1mg) sufficient for labeling up to 1mg of antibody Discover more at 1
4 INTRODUCTION 2. ASSAY SUMMARY Discover more at 2
5 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit as given in the table upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 6. Aliquot components in working volumes before storing at the recommended temperature. 5. LIMITATIONS Conjugation kit intended for research use only. Do not use kit or components if it has exceeded the expiration date on the kit labels. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. Discover more at 3
6 GENERAL INFORMATION 6. MATERIALS SUPPLIED Item 30 µg (3 x 10 µg) Amount 300 µg (3 x 100 µg) 1 mg (1 x 1 mg) Storage Condition Streptavidin 3x10 µg 3x100 µg 1x1 mg -20 C Mix Quencher 1 Vial 1 Vial 1 Vial -20 C Modifier 1 Vial 1 Vial 1 Vial -20 C Discover more at 4
7 GENERAL INFORMATION 7. MATERIALS REQUIERED NOT SUPPLIED These materials are not included in the kit, but will be required to successfully perform this assay: Unconjugated antibody or protein Microfuge tubes (0.5 or 1.5 ml) Microfuge Adjustable pipette 8. TECHNICAL HINTS Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Discover more at 5
8 PREPARATION 9. REAGENT PREPARATION Briefly centrifuge small vials at low speed prior to opening to avoid reagent loss in tube caps. 10. SAMPLE PREPARATION Pre-conjugation Considerations: The purified antibody to be labeled should ideally be in mm amine-free buffer (e.g. MES, MOPS, HEPES, PBS), ph range 6.5 to Common non-buffering salts (e.g. sodium chloride), chelating agents (e.g.edta), Borate buffer and sugars have no effect on conjugation efficiency. Azide (<0.1%), BSA (<0.1%) and Gelatin (<0.1%) have little or no effect. Glycerol up to 50% has no effect Avoid buffer components that are nucleophilic, as these may react with Conjugation Kit chemicals. Primary amines (e.g. amino acids, ethanolamine or Tris) and thiols (e.g. mercaptoethanol or DTT) fall within this class. Thimerosal (thiomersal), Merthiolate, Glycine and Proclin should also be avoided If intending to use this kit for immunohistochemistry, it is recommended that there be no gelatin or BSA present Recommended amount and volume of antibody for optimal results: Vial Size Amount of Antibody (µg) Volume of Antibody (µl) 10 µg µg mg Discover more at 6
9 PREPARATION Antibody concentrations of 1 mg/ml generally give optimal results. Antibodies with concentrations greater than 1 mg/ml should be diluted to 1mg/ml using PBS. Antibodies with concentrations less than 1 mg/ml can still be used to generate good conjugates, provided that the recommended volume of antibody is not exceeded. If the amount of antibody labelled is below the recommended amount, there may be unbound label post-conjugation. The excess label will be deactivated by the quencher and removed during the first wash step of any application. We recommend that antibodies below 0.5 mg/ml are concentrated to 1 mg/ml prior to use. Discover more at 7
10 PROCEDURE 11. CONJUGATION PROCEDURE Add 1 µl of Modifier to each 10 µl of antibody to be labelled, mix gently Remove cap from vial of Streptavidin Mix and pipette the antibody sample (with added Modifier) directly onto the lyophilized material. Resuspend gently by withdrawing and redispensing the liquid once or twice using a pipette Replace cap on the vial and leave standing for 3 hours at room temperature (20-25 C). Conjugations can also be set up and left overnight; longer incubation times have no negative effect on the conjugate After incubating for 3 hours (or more), add 1 µl of Quencher reagent for every 10 µl of antibody used and mix gently. The conjugate can be used after 30 minutes. No separation steps are necessary Storage at 4 C is recommended for any conjugate. A preservative may be desirable for long-term storage. Discover more at 8
11 12. NOTES Discover more at
12 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2016 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. Discover more at
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