Exosomes Immunocapture and Isolation Tools: Immunobeads

Transcription

1 Product Insert Exosomes Immunocapture and Isolation Tools: Immunobeads HBM-BOLF-##/##-# HBM-BOLC-##/##-# HBM-BTLF-##/##-# Overall Exosome capture from Human Biological fluids Overall Exosome capture from Cell culture supernatant Tumor-derived Exosome capture and enrichment from Human Biological fluids This product is for research use only. It is highly recommended to read this users guide in its entirety prior to using this product. Do not use this kit or its components beyond the indicated expiration date.

2 Table of Contents PRODUCT INFORMATION 3 PRODUCT DESCRIPTION 3 PRODUCT CONTENT 3 PROTOCOL 4 PROCEDURE 4 PROTEOMIC ANALYSIS 5 COMPATIBILITY WITH RNA ANALYSIS 6 TROUBLESHOOTING 7 APPENDIX A 8 SAMPLES PREPARATION (Plasma, Serum, Urine, Cell culture supernatant) 8 APPENDIX B 9 Related Products 9 TECHNICAL SUPPORT 10 Latex Beads Product Insert HansaBioMed 2

3 PRODUCT INFORMATION PRODUCT DESCRIPTION Overview About Exosomes About the latex beads Immunobeads display high capturing efficiency due to specific antibody coating and favorable interaction with biological vesicles (exosomes) in the sample. They allow the use of different sample s volume (0,1 ml up to 1 ml). Exosomes are small endosome derived lipid nanoparticles ( nm) actively secreted by exocytosis by most living cells. Exosome release occurs either constitutively or upon induction, under both normal and pathological conditions, in a dynamic, regulated and functionally relevant manner. Both amount and molecular composition of released exosomes depend on the state of a parent cell. This is one of major premises of employing exosomes for research and diagnostic purposes. Exosomes have been isolated from diverse cell lines (hematopoietic cells, tumor lines, primary cultures, virus infected cells) as well as from biological fluids in particular blood (e.g. serum and plasma from cancer patients) and other body fluids (bronchoalveolar lavage fluid, pleural effusions, synovial fluid, urine, amniotic fluid, semen, saliva etc). Exosomes have pleiotropic physiological and pathological functions and an emerging role in diverse pathological conditions such as cancer, infectious and neurodegenerative diseases. Latex immunobeads can be efficiently recovered after incubation with the sample using a standard top-bench centrifuge. Magnetic immunobeads are under development and will be available soon on HansaBioMed catalog. Beads-purified exosomes captured on immunobeads can be directly used for analysis of protein markers or for RNA content. PRODUCT CONTENT Type of Immunobeads available: Immunobeads for Overall Exosome capture from Human Biological fluids (plasma, serum and urine); Immunobeads for Overall Exosome capture from cell culture supernatant; Immunobeads for Tumor-derived Exosome capture from Human Biological fluids (plasma serum and urine). Different sizes of Immunobeads are available depending on the downstream analysis, for more details check out our catalog in Exosomes Immunocapturing and Isolation Tools part. Available size 0.4, 1 and 4 microns. Content Pre-coupled Latex Immunobeads 10 or 20 reactions Expiration date 8 months, store at 4 C HansaBioMed 3 Latex Beads Product Insert

4 PROTOCOL PROCEDURE Overview Immunobeads have been designed for selective exosome capture from biological fluids and cell culture supernatants. Hereby proposed protocol is applicable to all immunobeads presented in the catalog. Products are for research use only and is not intended for diagnostic use. Exosomes immunocapture 1. Add 10 µl of pre-coupled beads to 0.5 ml up to 1 ml of biological sample (plasma, urine or cell culture supernatant previously precleared in according with indications in appendix 1. Incubate overnight at 4 C in rotator. Remark: Incubation can be carried out also at room temperature for at least 4 hours in rotator. 2. After exosome binding wash beads 3 time with 500 µl of PBS or PBS + Tween 0.05% by resuspending up and down times. In each step remove the supernatant by centrifugation at 5000 g for The prepared beads can be used for further captured exosome characterization including both protein and nucleic acid content analysis, or exosomes can be recovered and studied. Exosome elution from beads (Only for 0.4 and 1 micron bead size, 10 and 20 reactions) 1. Add 10 μl of Exosome Recovery Buffer, vortex for 30, incubate at RT for 5. Vortex again 30 and add 40 μl of PBS 1X. 2. Centrifuge 10 at 5000 g, transfer the supernatant in a clean tube (low binding) and store in ice. 3. Repeat the elution step as indicated in step Centrifuge as indicated above. 5. Collect the two fractions of supernatant all together. Beads regeneration* 1. Add 500 μl of regeneration buffer, incubate for 5 at RT. 2. Centrifuge 10 at 5000 g, discard the supernatant. 3. Wash beads with 1 ml of PBS, centrifuge as indicated above, discard the supernatant. 4. Resuspend beads in 10 μl of PBS. * Beads can be reused for not more than twice. Latex Beads Product Insert HansaBioMed 4

5 PROTEOMIC ANALYSIS Exosomes incorporate a wide range of membranes and cytosolic proteins involved in many cellular functions and reflecting both their endosomal origin and a protein fingerprint unique for parent cell type and condition. Starting with exosomes capture on immunobeads is an ideal starting point for further studies on exosome biomarkers. Immunobeads for Overall Exosome capture from Biological fluids Alix 1, 3 and 5 2, 4 and 6 7 Detection of exosomes immunocaptured on beads with three different antibodies from 0,5 ml of unfractioned human plasma Ultracentrifuged human plasma after beads immunocapture Immunocapture with beads coated with exosome unrelated antibody 8 Ultracentrifuged exosomes from 0,5 ml of plasma 9 Ultracentrifuged exosomes from 1 ml of plasma 10 Ultracentrifuged exosomes from 2 ml of plasma Figure 1: Comparison between exosomes immunocaptured on beads and purified by ultracentrifugation from 0,5 ml of human healthy donor plasma. Intensity of the immunoreactive band detected with anti-alix antibody shows the advantages in the using of immunocapture techniques for exosomes isolation Immunobeads for Overall Exosome capture from Cell culture supernatant COLO1 Cell Supernatant Alix Empty Beads Immunobeads Figure 2: Exosomes from COLO1 cell supernatant captured on latex immunobeads analyzed by WB with antibody anti-alix Immunobeads for Tumor-derived Exosome capture and enrichment from Biological fluids Healthy donors Melanoma patients Alix Figure 3: Enrichment of tumor derived exosomes captured with latex immunobeads coupled with specific antibody against exosomal tumor antigen. Lane 2 and 5 performed with overall exosomes capture immunobeads, and lane 3 and 6 with tumoral derived exosomes capture immunobeads. HansaBioMed 5 Latex Beads Product Insert

6 COMPATIBILITY WITH RNA STUDIES Figure 2: Comparison of RNA yield obtained by extraction from ultracentrifuged exosomes from 0,8 ml of human plasma and immunocaptured on latex beads from 0,8 ml of human plasma Ultracentrifuge exosomes Beads immunocaptured exosomes #1 #2 #3 Blank Figure 3: RT-PCR analysis of total exosomal RNA derived from exosomes immunocaptured by 0,5 ml of three different human plasma samples β-actin Latex Beads Product Insert HansaBioMed 6

7 TROUBLESHOOTING Problem/Cause Low efficiency in immunobeads precipitation Immunobeads damage Suggested Solution Usage of low-binding tubes is recommended. Increase centrifugation time (15 or 20 at 5000 g). Not increase centrifugation speed over 5000 g. Conditions that would be damaging to the immunobeads include freezing (irreversible aggregation), high temperature (>95 C), exposure to organic solvents and high centrifugation speed (swelling, deformation, sticking of immunobeads) Presence of aggregates and hard immunobeads resuspension: Formation of small aggregates may happen collecting immunobeads after samples incubation. Small aggregates usually do not cause problems and can be resuspended by pipetting several time during washing steps. Incubation 5 at 37 C can help to disrupt small aggregates. Irreversible aggregation in precipitation phase Nonspecific absorption Exosomes markers detection in western blotting Irreversible aggregation of immunobeads after incubation with biological samples may happen if samples are not well precleared or too concentrate. Preclear plasma samples following protocol indicated in appendix 1 before incubation with immunobeads. Dilute concentrated urine or cell culture supernatants samples with PBS 1x or a solution of NaCl 0,9 M. Include or increase the percentage of Tween 20 in buffer for washing. Recommended concentrations: 0,05 % to 0,2% Beads are coupled with mouse or rabbit antibody for exosomes immunocapture. The signal of heavy and light immunoglobulins chains can appear during the detection phase if a conventional secondary antibody HRP conjugated is used. The use of detection primary antibodies HRP or biotin tagged eliminate this problem. HansaBioMed 7 Latex Beads Product Insert

8 APPENDIX A SAMPLE PREPARATION (Plasma, Serum, Urine, Cell culture supernatant) Protocol Plasma and serum samples preparation Prepare samples by 3 centrifugation steps to eliminate red blood cells and cellular debris: 10 at 300 g 20 at g 30 at g After each step save the supernatant. Processed plasma is ready to be loaded onto the plate (100 μl/well) or incubated with beads. Urine samples preparation Preclear urine samples by centrifugation at g for 20 at RT Filter by using 0.45 μm filter Concentrate urine samples by spin concentrator for 5-10 times for proteomic and times for nucleic acid studies*. Cell culture supernatant samples preparation Prepare cell supernatants by 3 centrifugation steps: 10 at 300 g 20 at g 30 at g Concentrate cell supernatant times in spin concentrator* *The quantity of exosomes could vary between samples. Concentration factors are given for information purposes only, a larger starting amount of sample should be used if the signal is weak. Latex Beads Product Insert HansaBioMed 8

9 APPENDIX B Related Products Overview As the first company entirely dedicated to Exosomes, we offer a panel of kits and reagents for Exosome Research. Ordering information on a variety of reagents and apparatus available from HansaBioMed is provided below. For more information, visit our website at Products Quantity Catalog Number ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from Biological fluids ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from human Serum ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from Cell culture supernatant ExoTEST Ready To Use Kit for Tumor-derived Exosome enrichment and quantification from Biological fluids Custommade ExoTEST for Specific Exosome capture and quantification Exosome Total RNA Extraction Kit (Immunobeads), 10 or 20 Reactions HBM-RTK-POF/## HBM-RTK-POS/## HBM-RTK-POC/## HBM-RTK-PTF/## HBM-RTK-CMK HBM-RNA-BOL-##/# Exosome Total RNA Extraction Kit (Immunoplate) HBM-RNA-POT/# Tumor-derived Exosome Total RNA Extraction Kit (Immunobeads), 10 or 20 Reactions HBM-RNA-BTL-##/# Tumor-derived Exosome Total RNA Extraction Kit (Immunoplate) HBM-RNA-PTT/# Lyophilized Exosomes from plasma (Healthy donor), from urine (Healthy donor) and from cell culture supernatant Exosome Binding Antibodies 2/4 or 6X 100μg 2/4 or 6X 30μg Check Exosome Standards available on Check Ab available on HansaBioMed 9 Latex Beads Product Insert

10 TECHNICAL SUPPORT Get support For the latest services and support information for all locations, go to At the website, you can: Search for user documents, handbooks, certificates of analysis, citations, and other product support documents. Access telephone and fax numbers to contact Technical Support and Sales facilities. Or contact us at Material Safety Data Sheet (MSDS) Material Safety Data Sheets (MSDSs) are available at Trademarks ExoTEST and HansaBioMed are trademarks of Hansabiomed OÜ..Other brands or product names are trademarks of their respective holders. Research Use All products are sold for research or laboratory use only and are not intended to be administered to humans or used for medical diagnostics. Latex Beads Product Insert HansaBioMed 10

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12 For the latest product and technical information, visit hansabiomed.eu and exotest.eu HansaBiomed Homepage Online Shop HansaBioMed LLC Akadeemia tee 15, Tallinn, ESTONIA Contact in US Galen Laboratory Supplies P.O. Box 682 Middletown, CT Tel: Fax: Tel: Fax: For support visit or Visit our website and