FastGlycoProteinTM Isolation Kit WGA Resin
Contents
1. 50 preps For use with all plant and animal samples e yse 50 100 mg tissue per 2mL tube e Lysis and isolation with single phase organic solution in less than 90 minutes FastGlycoProtein Isolation Kit WGA Resin FastRNA Pro Soil Direct Kit and FastRNA Pro Soil Indirect Kit 116070050 116075050 respectively 50 preps e Isolate RNA from soil samples direct kit and washed soil indirect kit in less than 2 hours e Variety of soil and environmental sample types e RNA protected during and after processing e Humic acids reduced to allow uninhibited RT PCR e Includes additional reagents for even further purification if necessary e SPIN filters streamline silica handling FastProtein Blue Matrix 116550400 50 preps 116550500 100 preps e Release of proteins from gram positive and gram negative bacteria in 40 seconds Protein extracts are ready for immediate electrophoresis or purification e deal for optimizing induction conditions FastProtein Red Matrix 116550600 50 preps 116550700 100 preps Release of proteins from yeast cells and fungi in 40 seconds Protein extracts are ready for immediate electrophoresis or purification deal for optimizing induction conditions Appendix 1 General recommendations for processing samples with FastPrep Instruments The fill volume in the lysing matrix tube after the addition of the Cell Lysis Solution to the sample should allow sufficient air space in the2. improve and shearing of existing genomic DNA may begin to occur Samples with 2 spheres should be processed carefully in order to balance increased yield and lysis against increased DNA shearing by varying speed and or time settings General Speed Time and Lysing Matrix Recommendations per Sample Type D D D A D A A A D D D A A D D A D A A D D D A D D A D D A D D D D E E E E E E E E E FastGlycoProtein Isolation Kit WGA Resin Application Manual FastGlycoProtein Isolation Kit WGA Resin Protocol Revision 116550900 13NOV Cat No 116550900 Worldwide Ordering and Technical Support USA Worldwide Headquarters Tel 800 854 0530 Fax 800 334 6999 Europe Phone 00800 7777 9999 Fax 00800 6666 8888 Australia Tel 61 2 9838 7422 Tel 1800 249 998 Fax 61 2 9838 7390 Fax 1800 818 671 Belgium Tel 32 2 466 00 00 Fax 32 2 466 26 42 Canada Tel 800 854 0530 Fax 800 334 6999 China Tel 86 21 6281 5005 Fax 86 21 6281 5663 France Tel 33 3 88 67 54 25 Fax 33 3 88 67 19 45 Germany Tel 0800 426 67 337 Fax 0800 629 67 337 India Tel 91 22 22631257 58 Fax 91 22 22631259 Japan Tel 81 03 3808 2102 Tel 0120 78 8020 Fax 81 03 3808 2401 Latin America Tel 1 440 337 1200 Fax 1 440 337 1180 The Netherlands Tel 0800 0227 416 Fax 0800 0227 489 New Zealand Tel 64 9 912 2460 Fax 64 9 838 4209 Poland Tel 48
3. 22 659 58 95 Fax 48 22 658 45 05 Russia Tel 7 495 661 0008 Fax 33 3 88 67 19 45 Serbia Tel 381 11 242 1972 Fax 381 11 242 1949 Singapore Tel 65 6775 0008 Fax 65 6774 6146 Switzerland Tel 41 61 271 0007 Fax 41 61 271 0084 United Kingdom Tel 0800 282 474 Fax 0800 614 735 mpbio com sampleprep support mpbio com MP Biomedicals e 29525 Fountain Parkway e Solon OH 44139 USA tel 800 854 0530 fax 800 334 6999
4. Application Manual FastGlycoProtein Isolation Kit WGA Resin Isolate glycoproteins from complex protein mixtures and other biological samples in less than 60 minutes Cat No and Size 116550900 10 preps su Call Storage Refrigerated 49C One Source World of Protocol Revision 116550900 13NOV Sample Prep Solutions MSDS available online mpbio com sampleprep FastGlycoProtein Isolation Kit WGA Resin TABLE OF CONTENTS 1 Introduction to the fastGlycoProtein Kit and the FastPrep systems cccccccsecsscscsscsesscsesecssesssesecsecsssecsssessesesatsessesasees 3 2 Kit Components Storage amp User Supplied Materials 4 2 1 FastGlycoProtein Kit components ss 4 AUCI 4 2 3 User Supplied Materials nnsnseneneneeeeeneneaenennenensrsrsrsrsinannnnnnnnnnrnrnrnrnrnnnnen 4 3 Important Considerations Before Use sennenne 4 3 1 Buffer Equilibration esses 4 3 2 Sample Preparation ss 5 4 Safety 8 etna natia tete HUU 5 SM MEE 5 6 Tro bleshooftilig uscire ete rte tete retenta 8 7 Example Data Glycoprotein Isolation from Human Serum and CHO Lysate Samples and Gel 8 8 Recommended Refere
5. EDTA or other metal chelators 3 2 End product compatibility Samples purified with this kit are compatible with 1 D gel electrophoresis and Total Protein Bradford type Assays Other downstream applications may require sample processing to remove incompatible substances in the Elution Buffer To quantify protein using a BCA type Assay desalt sample using a 5 ml desalting column For 2 D gel electrophoresis clean up sample using the precipitation or dialysis or other purification technique 3 3 Abundant Protein Removal Removing albumin and IgG from serum samples improves isolation of less abundant glycoproteins for mass spec applications 4 Safety Precautions Some of the supplied kit reagents contain components that when in contact with human tissue may cause irritation Wear personal protective equipment to prevent contact with the skin or mucus membranes gloves lab coat and eye protection at all times when using this product Consult the Material Safety Data Sheet at www mpbio com for additional details This product is for research purposes only 5 Protocol IMPORTANT NOTE BEFORE STARTING PROTOCOL Equilibrate the Binding Wash and Elution Buffers to room temperature Sample Preparation 1 For serum samples or biological lysates from sonication douncing or other chemical digest dilute sample containing 1 to 1 5 mg of total protein 4 1 with 5X Binding Wash Buffer stock solution e g mix approximate 400 HL of vo
6. NA and proteins e Integrity and size of DNA RNA and proteins are retained e Nucleic acids and proteins are ready to use in downstream application FastDNA Kits FastDNA Kit and FastDNA Spin Kit 116540400 116540600 respectively 100 preps e Lyse and isolate DNA in less than 30 minutes e Plant animal yeast fungal and microbial samples No hazardous organic reagents required e SPIN filters streamline silica handling FastDNA Spin Kit FastDNA Spin Kit for Feces 116570200 50 preps e Lyse and isolate PCR ready DNA in less than 30 minutes e Variety of stool sample types No hazardous organic reagents required e SPIN filters streamline silica handling FastDNA 50ml Spin Kit for Soil 116560600 10 preps Process low microbial containing soil samples e Lyse and isolate PCR ready DNA in less than 60 minutes Variety of soil and environmental sample types No hazardous organic reagents required e SPIN filters streamline silica handling FastRNA Pro Blue Kit 116025050 50 preps e For use with gram positive and gram negative bacteria e Lyse up to 1010 cells per 2mL tube e Lysis and isolation with single phase organic solution in less than 90 minutes FastRNA Pro Red Kit 116035050 50 preps For use with yeast cells and fungal tissue e Lyse up to 1010 cells per 2mL tube e Lysis and isolation with single phase organic solution in less than 90 minutes FastRNA Pro Green Kit 116045050
7. comparison of kits using WGA resin CHO lysate and human serum samples were processed with the FastGlycoProtein Isolation Kit WGA Resin and with other commercially available WGA resins An equivalent amount of total protein was applied to each resin Eluted glycoprotein fractions were normalized by volume and resolved on 8 16 polyacrylamide gels a Eluted glycoprotein fraction from applied CHO lysate b Eluted glycoprotein fraction from applied human serum 8 Recommended Reference Format for Publications Glycoproteins were isolated from specific sample using the FastGlycoProtein Isola tion Kit WGA Resin and the FastPrep insert model Instrument MP Biomedicals Santa Ana CA 9 References Cooper C A et al 2001 GlycoSuiteDB A new curated relational database of glycoprotein glycan structures and their biological sources Nucl Acid Res 20 1 332 5 Cummings R D 1997 Affinity chromatography of oligosaccharides and glycopeptides Affinity Separations A Practical Approach Matejschuk P Ed Oxford Univ Press London pp 123 139 Ghosh D et al 2004 Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins J Proteome Res 3 841 850 Young N M et al 2002 Structure of the N linked glycan present on multiple glycoproteins in the gram negative bacterium Campylobacter jejuni J Biol Chem 277 42530 9 10 Technical Support For technical support with this pro
8. duct please contact our MP Biomedical s Technical Support Team at 1 800 854 0530 by email at biotech mpbio com or visit us online at www mpbio com for live support 11 Related Products Description Size Catalog Coomassie Brilliant Blue R250 1104821636 Ammonium Sulfate Ultra Pure 1104808211 Biotrans PVDF Transfer Membranes FastPrep 96 Instrument 116010500 FastPrep 24 Instrument 116004500 FastDNA Kit 100 preps 116540400 FastDNA SPIN Kit 100 preps 116540600 FastDNA SPIN Kit for Soil 50 preps 116560200 FastDNA 50ml SPIN Kit for Soil 10 preps 116570200 FastRNA Pro Soil Direct Kit 50 preps 116070050 FastRNA Pro Soil Indirect Kit 50 preps 116075050 FastRNA Pro Red Kit Yeast amp Fungus 50 preps 116035050 FastRNA Pro Green Kit Plant amp Animal 50 preps 116045050 FastRNA Pro Blue Kit Bacteria 50 preps 116025050 FastProtein Blue Matrix 50 preps 116550400 FastProtein Red Matrix 50 preps 116550600 FastGlycoProtein Isolation Kit WGA Resin 12 Product Use Limitation amp Warranty The products presented in this instruction manual are for research or manufacturing use only They are not to be used as drugs or medical devices in order to diagnose mitigate treat or prevent diseases in humans or animals either as part of an accepted course of therapy or in experimental clinical investigation These products are not to be used as food food additives or general household it
9. ems Purchase of MP Biomedicals products does not grant rights to reproduce modify or repackage the products or any derivative thereof to third parties MP Biomedicals makes no warranty of any kind expressed or implied including merchantability or fitness for any particu lar purpose except that the products sold will meet our specifications at the time of delivery Buyer s exclusive remedy and the sole liability of MP Biomedicals hereunder shall be limited to at our discretion no replacement or compensation product credits refund of the purchase price of or the replacement of materials that do not meet our specification By acceptance of the product Buyer indemnifies and holds MP Biomedicals harmless against and assumes all liability for the consequence of its use or misuse by the Buyer its employees or others including but not limited to the cost of handling Said refund or replacement is conditioned on Buyer notifying within thirty 30 days of receipt of product Failure of Buyer to give said notice within thirty 30 days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material s Gene Clean and FastPrep are registered trademarks of MP Biomedicals LLC Ready to use Protocols For DNA RNA And Protein Isolation From Any Sample Rapid and reproducible sample lysis and purification process No cross contamination with the closed lysing matrix tubes Increased yields of high quality DNA R
10. lume of sample with 100 HL 5X Binding Wash Buffer The total volume including dilution must not exceed 800 pL FastGlycoProtein Isolation Kit WGA Resin Optional For mechanical cell lysis using FastPrep instruments place the sample containing up to 1 5mg of proteins in a Lysing Matrix A tube Dilute sample 4 1 with 5X Binding Wash Buffer stock solution e g mix approximate 400 of volume of sample with 100 uL 5X Binding Wash Buffer The total volume including dilution must not exceed 800 Process sample in a FastPrep in accordance to the recommended set tings for the sample type see Appendix 1 Isolation of Glycoproteins 1 Prepare the 1X Binding Wash Buffer dilute 460 uL 5X Binding Wash Buffer with 1 840 uL of ultrapure water This is sufficient volume to process one sample 2 Inserta column into a collection tube 3 Gently swirl the bottle of WGA Lectin Resin to obtain a homogeneous suspension Use a wide bore or cut pipette tip to transfer 200 uL of 50 resin slurry to the column 4 Centrifuge 1 minute at 1 000 xg and discard the storage buffer Reuse the catch tube through step 12 5 Replace the column in the same catch tube and add 200 uL 1X Binding Wash Buffer to the resin Close the top cap and centrifuge column for 1 minute at 1 000 xg and discard rinse Repeat this step two times 3x washes are necessary to condition the resin 6 Place bottom cap on column and add sample from Sample Preparatio
11. n step total sample volume not to exceed 800 uL to the resin Close the top cap 7 Incubate column for 10 minutes at room temperature with end over end mixing using a rotator e g Labquake Shaker by Thermolyne Alternatively rock back and forth on a rocking platform 8 Remove top cap and then bottom cap from column Place column in the catch tube and replace top Note Remove top cap first and then quickly remove bottom cap and place im mediately in catch tube in order to prevent sample from leaking from the bottom of the column 9 Centrifuge column for 1 minute at 1 000 xg and discard flow through Note f desired save flow through for SDS PAGE or protein assay analysis as this will contain all other non glycosylated proteins if present 10 Reinsert column and add 400 uL 1X Binding Wash Buffer to the resin Cap column and centrifuge for 1 minute at 1 000 x g and discard flow through Repeat this step Place bottom cap on column and add 400 ul 1X Binding Wash Buffer to the resin Cap column and incubate for 5 minutes at room temperature with end over end mixing using a rotator Remove top cap and then bottom cap from column Place column in the collection tube and replace top cap Centrifuge column for 1 minute at 1 000 x g and discard rinse Repeat Steps 11 and 12 Replace bottom cap on column Add 200 yl Elution Buffer to resin and cap column Incubate column for 5 minutes at room temperat
12. nce Format for 8 MEI CIS EUER 9 10 Technical GUppOrt 5 cec A A died DF 9 11 Related Products sess 9 12 Product Use Limitation amp Warranty seen 10 19 Appendix tte ten tne to A OR Ge MEE LUCES 12 General Recommendations for FastPrep Processing Recommended settings by sample 1 13 2 mpbio com 1 Introduction to the FastGlycoProtein Isolation Kit WGA Resin and the FastPrep Systems The MP Biomedicals FastGlycoProtein Isolation Kit isolates glycoproteins from complex protein mixtures including serum and cultured cells as well as animal and plant tissue microbe and insect lysates using the lectin wheat germ agglutinin WGA immobilized on agarose Lectins are proteins that have a selective affinity for carbohydrate moieties The WGA lectin preferentially binds N acetyl glucosamine GICNAC and terminal GICNAC structures that are commonly present in many serum and membrane glycoproteins WGA also has affinity for sialic acid Protein glycosylation is a common post translational modification Asparagine N linked and serine threonine residues O linked are glycosylated during passage through the endoplasmic reticulum and golgi apparatus in eukaryotic and prokaryotic i e Archaea and Eubacteria systems Glycoconjugates are important for immune reg
13. sample tube for efficient FastPrep Instrument processing MP Biomedicals recommends using 100 200 mg of starting material as long as there is between 250 500 of empty space in the tube Sample loss or tube failure may result from overfilling the matrix tube The matrix tube caps must be secure but not over tightened to prevent sample leakage If the sample is too large for processing in a single tube divide the sample and process using multiple tubes MP Biomedicals Lysing Matrix particles and tubes have been rigorously tested and vali dated in the FastPrep Instrument The use of other products with the FastPrep Instru ment is not recommended and may result in sample loss or instrument failure A single 40 second run at a speed setting of 6 0 in the FastPrep Instrument is sufficient to lyse almost all samples If the user experimentally determines that additional processing time is required the sample should be incubated on ice in the Lysing Matrix A tube for at least 2 minutes between successive FastPrep Instrument homogenizations to prevent overheating the sample and tube MP Biomedicals recommends that all researchers begin the protocol with the Lysing Matrix A as supplied in the kit garnet matrix and single sphere If lysis is inefficient even after multiple runs of 40 seconds an additional inch ceramic sphere provided can be added on top of the sample Depending on the sample lysis and or yield may or may not
14. sing matrix particles are designed to efficiently lyse organisms such as plant and animal tis Sue cultured cells bacteria yeast and fungi including historically difficult sources such as eubacterial spores and endospores algae and nematodes while in the presence of a FastGlycoProtein Isolation Kit WGA Resin specially formulated lysis solution The FastPrep Instruments lyse samples quickly and efficiently most in 40 seconds or less all while preserving the protein s functionality and biological activity 2 Kit Components Storage and User Supplied Materials 2 1 FastGlycoProtein Isolation Kit Components Lysing Matrix A 10 x2 mL Tubes WGA Lectin Resin 1 1 mL settled as slurry Binding Wash Buffer 5X solution 6 5 mL Elution Solution 5 mL Spin Filter with caps 10 each Catch Tubes 20 each User manual 1 each MSDS Online www mpbio com 1 each Certificate of Analysis 1 each 2 2 Storage All FastGlycoProtein Isolation Kit ConA Resin should be stored at 4 C immediately upon receipt The Kit is shipped ambient The kit reagents are guaranteed for up to one year from the date of purchase of the kit 2 3 User Supplied Materials FastPrep Instrument optional Benchtop microcentrifuge capable of 10 000 xg rcf Inverting lab shaker or rocker Sterile water 3 Important Considerations Before Use 3 1 Proteases metal chelators If needed add protease inhibitors to samples however avoid cocktails containing
15. ulation inflam mation cell to cell adhesion and contact inhibition cell signaling protection against pro teolytic degradation and other biological processes This kit is easy to use and contains all the necessary components for isolating sialic acid GIcNAC containing glycans A sample containing up to 1 5 mg of total protein is first placed in lysing matrix A tube with the Binding Wash Buffer applied to it After the tissue cell lysis is complete the sample is transferred and applied to the ConA resin bed Following incubation the resin is washed and the bound glycoproteins are eluted Glycoproteins have been successfully isolated from serum as well as HeLa and CHO cell lysates in approximately 40 minutes The FastPrep Sample Preparation Instruments including the FastPrep 24 and Fast Prep 96 are high throughput homogenizers developed to disrupt thoroughly any tis sue or cells through the simultaneous bead beating and impaction of specialized Lys ing Matrix beads on the sample material The FastPrep Instruments use high powered complex motions to provide extremely quick and highly reproducible homogenization that surpasses early generation homogenizers as well as traditional extraction methods using enzymatic digestion sonication blending douncing or vortexing Samples are placed into a variety Lysing Matrix Vessel formats containing custom lysing matrix particles designed for optimized disruption of specific sample types The ly
16. ure with end over end mixing using a rotator Remove top cap and then bottom cap from column Place column in a new catch Replace top cap and centrifuge column for 1 minute at 1 000 x g Carefully set aside the catch tube and remove top cap Repeat Steps 14 16 Pool eluate 2 in the same catch tube that contains the first elute Store eluted glycoproteins on ice for immediate use or freeze for later analysis FastGlycoProtein Isolation Kit WGA Resin 6 Troubleshooting Problem Possible Cause Solution Low glycoprotein recovery in elution fraction Some glycoproteins have high affinity for the immobilized WGA and will not elute with Elution Buffer Increase incubation with elution buffer to 10 minutes or boil resin in 200 ul of SDS PAGE sample buffer for 5 minutes and then centrifuge column in a 2 ml tube for 1 minute at 1 000 x g to collect eluate Note Boiling the resin results in detachment of some lectin and also may release non specifically bound proteins Glycoprotein is not binding to the resin Sample contains metal chelator s Confirm EDTA or other metal chelators are not present in the sample 7 Example Data Glycoprotein Isolation from Human Serum and CHO Lysate Samples and Gel Electrophoresis Human Serum Supplier CHO Lysate Pierce m Supplier A Glycoprotein isolation from human serum and cell lysate performance
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