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User Guide HuProt™ Human Proteome Microarray v2.0 User

1. Cover slips e g LifterSlip by Thermo Scientific cat no 25X60124789001LS and Grace Bio Labs HS6024 hybrislip 6 0X24MM Fisher Scientific cat no NC9296662 Humidification chamber for low incubation volumes between 100 300 ul or a modi fied empty yellow tip box Additional Materials and Equipment for Data Analysis Microarray scanner e g Molecular Devices GenePix 4000B and computer Microarray analysis program e g GenePix Pro 6 1 General Reagents Bovine Serum Albumin IgG Free Protease Free Jackson ImmunoResearch Labo ratories KCI NaCl Tris base Tween 20 EN fl ep Page 6 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 7 TABLE OF CONTENTS ASSAYS Antibody Specificity 7 For research use only Not for use in diagnostic procedures Assays Using the HuProt Human Proteome Microarray I Monoclonal Antibody Specificity Determination Assay 1 1 1 2 Storage Store HuProt microarrays inside closed plastic slide holders at 80 C or ona layer of dry ice right until the blocking step The active surface of the HuProt microarray is the surface with the barcode IMPORTANT Itis critical to keep the HuProt microarrays ultra cold and ultra dry prior to use Do not let liquid condense onto the microarray surface before use Blocking Add 3 0 ml of blocking solution 5 BSA TBS T to each compartment of the 4 well plates Carefull
2. IMPORTANT If fluorescent probes are used at any point light exposure which may quench fluorescence must be minimized Cover all 4 well plates or other containers holding the microarrays or labeled probes with aluminum foil or use a lightproof storage container Scan and Store the Microarrays e After assays are completed scan the microarrays immediately highly preferred or store them in a lightproof microscope slide box at 20 C During storage the active surface of the microarray should not touch any other surfaces including other microarrays IMPORTANT Microarrays must be scanned within 3 days after the assay is performed Me fl ep Page 4 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 5 TABLE OF CONTENTS SUMMARY 1 Additional Reagents and Materials 5 For research use only Not for use in diagnostic procedures Additional Reagents and Materials Materials Secondary Antibodies for Detection Step e The quality of commercially available secondary antibodies varies widely Please test all secondary antibodies thoroughly before conducting your assay the second ary antibodies listed in this manual are provided as examples e Create grid alignment for data analysis using the GST tagged proteins printed on the HuProt microarray as a visual reference Carry out anti GST probing after the pri mary assay has been conducted on the microarray and the data
3. Repeat for a total of three 10 min washes Do not let the HuProt microar ray dry out at any time IMPORTANT For all steps below cover the 4 well plates containing the Hu Prot microarrays tightly with aluminum foil during all incubations and washes to minimize light exposure Page 11 TABLE OF CONTENTS II 6 ASSAYS Serum Profiling 10 II 7 e e 11 8 For research use only Detection Add Secondary Antibodies Dilute the secondary antibody in blocking buffer per manufacturer s direc tions for Western Blot use Add 3 0 ml of the diluted secondary antibody to each freshly washed HuProt microarray Cover the 4 well plate with aluminum foil and incubate for 1 1 5 hrs with gentle shaking at room tem perature Washing Remove the buffer containing secondary antibodies by aspiration Add 4 0 ml TBS T to briefly rinse the microarray repeat for a total of three short rinses Add 4 0 ml of TBS T and incubate for 10 min at room temperature with gentle shaking Remove the wash buffer from a corner of the 4 well plate by aspiration Repeat for a total of three 10 min washes Briefly rinse the slides three times with ddH O Drying Place clean room wipes or paper towels on the bottom of a black micro scope slide box will hold several microarrays or prepare plastic conical 50 ml tubes each will hold one microarray EN fl py Not for use in diagnostic procedures Pa ge 1 1 NEXTGEN PROTEOMICS CDI Laboratories
4. User Guide e Remove the HuProt microarray from the 4 well plates and tap the edge lightly on a clean room wipe or paper towel to remove ex HuProt cess fluid Do not touch the active surface of the microarray Care Human Prote me Mictoarray v2 0 fully slot the microarrays into the microscope slide box the mi croarrays will be perpendicular to the paper towels lining the Page 12 box If you are using 50 ml tubes carefully slide a single micro array lengthwise into the tube Spin the microscope slide box or the 50 ml tubes containing the microar rays in a centrifuge at 800 rpm for 3 min to remove excess fluid spinning at higher speeds may break the microarrays After centrifugation carefully remove the HuProt microarrays and discard the clean room wipe or pa per towel TABLE OF CONTENTS 11 9 Scanning e The HuProt microarray can be scanned immediately highly preferred or it can be stored at 20 C in a lightproof box ASSAYS IMPORTANT Microarrays must be scanned within 3 days after the assay is performed Serum Profiling 10 Appendix for Serum Profiling Assay Recipes e TBS T Buffer TBS pH 7 5 0 1 Tween 20 see General Appendix buffer recipe Blocking Solution 5 BSA w v in TBS T Reference Hu CJ et al 2012 Identification of new auto antigens for primary biliary cirrhosis using human proteome microarrays Mol Cell Pro teomics 11 9 669 80 ee ee NZ eee HICDI For
5. 2 mM MgCl 5 mg ml BSA in SuperBlock T20 PBS Blocking Buffer Thermo Scientific Cat No 37516 RNA binding buffer 2 mM MgCl 2 mg ml BSA 10 ug ml ssDNA herring sperm in 1X PBS lt lt am Page 15 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 16 TABLE OF CONTENTS ASSAYS DNA Binding 16 For research use only Not for use in diagnostic procedures Assays Using the HuProt Human Proteome Microarray IV IV 1 IV 2 IV 3 IV 4 Fluorescently Labeled DNA Binding Assay Storage Store HuProt microarrays in closed plastic slide holders at 80 C or on a layer of dry ice right until the blocking step The active surface of the HuProt microarray is the surface with the barcode IMPORTANT It is critical to keep the HuProt microarrays in a cold dry environment and to prevent liquid from condensing onto the microarray sur face before use Blocking Place the HuProt microarrays in a humidification chamber Carefully pipette 200 yl of DNA hybridization buffer pre chilled on ice onto the active surface of each HuProt microarray Do not let the pipette tip touch the microarray surface Cover the microarray with a glass cover slip to prevent evaporation Cover the humidification chamber with aluminum foil to minimize exposure to light Incubate with gentle shaking for 3 hrs at 4 C NOTE If you do not have a humidification chamber
6. 3 ug protein in 3 0 ml blocking solution to a 4 well plate Immerse one blocked HuProt microarray into each compartment with the active side up Cover the plates with aluminum foil to minimize light exposure which may quench the fluo rescence Incubate with gentle shaking for 1 hr at room temperature Low volume assay If a limited amount of labeled protein is available carefully pipette the diluted sample 200 ng protein in 200 ul blocking np fl py Page 19 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 20 TABLE OF CONTENTS V 5 ASSAYS Protein Binding 19 solution onto the active surface of the blocked HuProt microarray Be careful not to let the pipette tip touch the microarray surface Cover the microarray with a fresh cover slip Place the coverslipped microarrays in a humidification chamber to minimize evaporation NOTE If you do not have a humidification chamber you can adapt an empty yellow tip box for this purpose Place clean room wipes or wet paper towels at the base of the yellow tip box and replace the empty tip rack on top of the towels Carefully place up to four coverslipped HuProt mi croarrays on the rack and then close the lid Cover the humidification chamber with aluminum foil to minimize light ex posure and incubate with gentle shaking at room temperature for 1 hr Washing High volume assay For reactions using 3 0 ml of dilut
7. is to float the cover slip off by immersing the microarray in a larger volume of wash buffer Rinse each slide briefly with 4 0 ml of TBS T completely removing the buffer after each wash by aspiration Repeat for a total of three short washes Add 4 0 ml of TBS T and incubate with gentle shaking at room temperature for 10 min then remove the buffer by aspiration Repeat for a total of three washes Detection Add Secondary Antibodies Dilute secondary detection antibody in blocking buffer to the manufacturer rec ommended concentration Add 3 0 ml of diluted secondary antibody to each compartment in a 4 well plate containing the HuProt microarrays Cover the 4 well plates with aluminum foil and incubate at room temperature for 1 hr with gentle shaking np fl py NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 9 TABLE OF CONTENTS ASSAYS Antibody Specificity 7 Reference Jeong JS et al 2012 Rapid identification of monospecific monoclonal antibodies using a human proteome microarray Mol Cell Proteomics 11 10 1074 mcp 0111 016253 For research use only Not for use in diagnostic procedures e Remove the buffer containing the secondary antibody from a corner of the 4 well plate by aspiration IMPORTANT After the secondary antibody is added store the microarrays in the dark For all incubation and washing steps below cover the 4 well plates contain in
8. a Human Proteome Microarray and the Construction of an OGT In teractome Proteomics 14 9 1020 30 doi 10 1002 pmic 201300144 Epub 2014 Mar 25 Ma TM et al 2014 Serine Racemase Regulated by Binding to Stargazin and PSD 95 Potential NUDA AMPA Glutamate Neurotransmission Cross talk J Biol Chem pii joc M114 571604 Epub ahead of print First Published on August 27 2014 Page 23 y CDI NEXTGEN PROTEOMICS e e eyes tet a NEXTGEN PROTEOMICS Guanajibo Research and Innovation Park 4005 St B Road 114 Km 1 3 Mayaguez PR 00682 T 787 806 4100 Ext 233 F 787 806 4006 www cdi lab com
9. research use only Not for use in diagnostic procedures Pa ge 1 2 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 13 TABLE OF CONTENTS ASSAYS RNA Binding 13 For research use only Not for use in diagnostic procedures Assays Using the HuProt Human Proteome Microarray 11 4 Fluorescently Labeled RNA Binding Assay Storage Store HuProt microarrays in plastic slide holders at 80 C or on a layer of dry ice right until the blocking step The active surface of the HuProt microarray is the surface with the barcode IMPORTANT Itis critical to keep the HuProt microarrays ultra cold and ultra dry prior to use Do not let liquid condense onto the microarray surface before use Ill 2 Ill 3 Ill 4 Blocking Add 3 0 ml of blocking solution to each compartment in the 4 well plates Carefully use fine nosed tweezers to remove one microarray from the plastic slide holder that is resting on dry ice Immediately submerge the HuProt microarray active side up in a compartment containing blocking buffer Incubate with gentle shaking for 5 min Carefully pour off the block ing buffer or remove it by aspiration Add 3 0 ml fresh blocking buffer to the compartment in the 4 well plate and incubate the microarray with gentle shaking for 1 5 2 hrs at room temperature Sample preparation low volume assay Dilute the fluorescently labeled RNA sample to 250 nM i
10. scanned using an anti Glutathione S Transferase antibody e g Glutathione S Transferase S japoni cum form EMD Millipore cat no A 21428 Next select a secondary antibody with a detection wavelength that is different from the wavelength used to detect the primary reaction NOTE The GST signals obtained after your primary assay should not be used as a measure of the HuProt microarray quality as all proteins on the microarray will become inactive after the primary assay If too much anti Glutathione S Transferase antibody is added this may result in a very high background and may negatively affect data analysis Additional materials and equipment for incubations assays Aluminum foil Automatic pipettes Cleanroom wipes preferred or paper towels Micropipettes Orbital shaker Sterile disposable micropipette tips Sterile serological pipettes Fine nosed tweezers Vacuum system Vortex Plastic 4 well plates to store HuProt microarrays during the blocking reaction and washing steps e g Thermo Scientific Nunc Dishes Rectangular 4 Well or Fisher Scientific No 12 565 495 np fl ep Page 5 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 6 TABLE OF CONTENTS SUMMARY 1 Additional Reagents and Materials 5 For research use only Not for use in diagnostic procedures BD Shoulder Cover Microscope Slide Box lightproof e g Fisher Scientific cat no 22 167 403
11. CDI Laboratories User Guide TABLE OF CONTENTS SUMMARY 1 Storage and Handling 2 Overview Key Steps for HuProt Microarray Use 3 Additional Reagents and Materials 5 ASSAYS Antibody Specificity 7 Serum Profiling 10 RNA Binding 13 DNA Binding 16 Protein Binding 19 APPENDIX Recipes 22 HuProt Literature Citations 23 For research use only Not for use in diagnostic procedures HuProt Human Proteome Microarray v2 0 CDI Laboratories T 787 806 4100 Ext 233 Guanajibo Research and Innovation Park F 787 806 4006 4005 St B Road 114 Km 1 3 supportfacdi lab com Mayaguez PR 00682 www cdi lab com Summary The HuProt human proteome microarray provides the largest number of unique human proteins known to be included on a single slide allowing thousands of interactions to be profiled in high throughput Microarray type Functional protein microarray Species Human Slide type Coated glass Detection method Fluorescence Number of proteins 19 394 The HuProt human proteome microarray is available on a variety of glass surfaces including FAST FullMoon SuperEpoxy SuperAldehyde SuperNHS Ni NTA PATH and Schott The HuProt version 2 0 microarray contains 19 394 unique proteins 19 275 in dividually purified human and 119 mouse proteins This content encompasses 15 275 unique human genes and 119 unique mouse gene symbols Recombinant proteins are expressed in the yeast S cere
12. Guide HuProt Human Proteome Microarray v2 0 Page 3 TABLE OF CONTENTS SUMMARY 1 Overview Key Steps for HuProt Microarray Use 3 For research use only Not for use in diagnostic procedures Overview Key Steps for HuProt Microarray Use Prepare a cold environment It is critical to keep the microarrays ultra cold and ultra dry right until they are used HuProt microarrays should be stored at 80 C Before removing HuProt microar rays from storage place a layer of dry ice pellets inside a styrofoam box with a lid and cover tightly Remove a plastic slide holder containing HuProt microarrays from storage at 80 C and place it lengthwise on top of the dry ice Add an additional layer of dry ice pellets or a sheet of frozen gel coolant on top of the plastic slide holder Prepare 4 well plates for the blocking step 4 well plates are used to hold the HuProt microarrays during the blocking reaction in the case of high volume samples and washing steps e g Nunc 4 well rect angular dishes Fisher Scientific No 12 565 495 Each compartment of the 4 well plates is used to hold one microarray To minimize cross contamination of samples during the reaction step some users choose to keep one compartment empty in between microarrays Add 3 0 ml of blocking buffer to each compartment of the 4 well plates e g Nunc 4 well rectangular dishes Fisher Scientific No 12 565 495 IMPORTANT Handle the microar
13. array face up in the 3 0 ml of diluted test sample no coverslip is needed e For low volume assays 100 300 ul evaporation must be minimized Add the sample to the active surface of the microarray and carefully cover the microarray with a new cover slip Place the covered microarray in a humidification chamber NOTE If you do not have a humidification chamber an empty yellow tip box may be adapted to this purpose Place wet clean room wipes or paper towels in the base of the yellow tip box and replace the empty tip rack above them Carefully place up to four coverslipped HuProt microarrays on the rack and then close the lid Washing e When assays are completed the microarrays are washed in 4 well plates For low volume reactions carried out in a humidification chamber the cover slip must first be removed without touching or scratching the microarray surface Immerse the covered microarray in a compartment of a 4 well plate into which 4 0 ml of wash buffer has previously been added the buffer will vary depending on the assay Us ing fine nosed tweezers carefully lift off the cover slip starting from the barcoded end of the microarray Do not touch or scratch the active surface of the microarray An alternative is to float the cover slip off by immersing the microarray in a larger volume of wash buffer e For high volume samples the washes may be carried out in the same 4 well plates in which assays and blocking were conducted
14. cation of proteins that interact with alpha A crystallin using a human proteome microarray Mol Vis 20 117 124 eCollection 2014 Deng RP et al 2014 Global Identification of O GlcNAc Transferase OGT Interactors by a Human Proteome Microarray and the Construc tion of an OGT Interactome Proteomics 2014 14 9 1020 30 doi 10 1002 pmic 201300144 Epub 2014 Mar 25 Ma TM et al 2014 Serine Racemase Regulated by Binding to Stargazin and PSD 95 Potential NMDA AMPA Glutamate Neu rotransmission Cross talk J Biol Chem pii jbc M114 571604 Epub ahead of print First Published on August 27 2014 For research use only CD Not for use in diagnostic procedures Pa ge 21 NEXTGEN PROTEOMICS e e eyes CDI Laboratories User Guide General Appendix HuProt Buffers Human Proteome Microarray v2 0 TBS Buffer Recipe 1X Page 22 e 50 mM Tris Cl pH 7 5 150 mM NaCl TABLE OF CONTENTS e 10X TBS For 1 liter of 10X TBS stock buffer dissolve the following in 800 ml of distilled water 60 5 g Tris 87 6 g NaCl e Adjust pH to 7 5 and add distilled water to 1L Sterilize by autoclaving or by filtration and store at room temperature TBS T e Add 1 0 ml Tween 20 to 100 ml 10X TBS solution Make up to 1 liter with distilled water Stir until homogenous Tween 20 is very viscous and may stick to pipette tips A 10 solution is easier to dispense than the undiluted form Phosphate Buffered Saline PBS Rec
15. cover slip to minimize evaporation Cover the humidification chamber with aluminum foil to minimize exposure to light Incubate with gentle shaking overnight at 4 C Washing One wash cycle is sufficient for this assay Immerse the coverslipped mi croarray in 4 0 ml of pre chilled wash buffer in a compartment of a 4 well plate Carefully remove the cover slip using fine nosed tweezers taking care not to scratch the active surface alternatively float the cover slip off by immersing the microarray in a larger volume of wash buffer Incubate with gentle shaking for 1 3 min at at 4 C Remove the HuProt microarrays from the wash buffer and tap the edge lightly on a clean room wipe or paper towel to remove excess buffer Do not touch the active surface of the microarray Drying Place clean room wipes or paper towels on the bottom of a black micro scope slide box will hold several microarrays or prepare plastic conical 50 ml tubes each will hold one microarray Remove the HuProt microarray from the 4 well plates and tap the edge lightly on a clean room wipe to remove excess fluid Do not touch the active surface of the microarray Carefully slot the microarrays into the microscope slide box placing the microarrays perpendicular to the paper towels lining the base of the box If you are using 50 ml tubes carefully slide a single microarray lengthwise into the tube Spin the microscope slide box or the 50 ml tubes containing th
16. e microar rays in a centrifuge at 800 rpm for 3 min to remove excess fluid spinning at higher speeds may break the microarray After centrifugation carefully remove the HuProt microarrays and discard the clean room wipes EN fl ep Page 17 NEXTGEN PROTEOMICS CDI Laboratories User Guide IV 7 Scanning and Storage e The HuProt microarrays can be scanned immediately preferred or HuPror stored at 20 C in a lightproof box Human Proteome Microarray v2 0 IMPORTANT Microarrays must be scanned within 3 days after the assay is Page 18 performed TABLE OF CONTENTS _ Appendix for Fluorescently Labeled DNA Binding Assay Recipes e DNA hybridization buffer store on ice or at 4 C 25 mM HEPES at pH 8 0 50 mM potassium glutamate 0 1 Triton X 100 8 mM magnesium acetate 3 mM DTT freshly added to buffer before use 4 uM poly dA dT 10 glycerol e Wash buffer Same as above but without 4 uM poly dA dT ASSAYS DNA Binding 16 nf Reference Hu S et al 2009 Profiling the human pro tein DNA interactome reveals ERK2 as a transcriptional repressor of interferon signal ing Cell 139 3 610 22 ae co For research use only Not for use in diagnostic procedures Pa ge 18 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 19 TABLE OF CONTENTS ASSAYS Protein Binding 19 For research use only Not for use in diagnostic procedures Assays U
17. ed test protein remove the reaction buffer from a corner of the 4 well plate by aspiration Add 4 0 ml PBS T to briefly rinse the microarray and remove the buffer by aspiration Repeat for a total of three short washes Low volume assay For reactions using a low volume of diluted test pro tein 200 pl add 4 0 ml PBS T to each compartment of a 4 well plate Submerge the coverslipped HuProt microarray in a compartment and carefully remove the cover slip using fine nosed tweezers being careful not to touch the active surface of the microarray alternatively float the cover slip off by immersing the microarray in a larger volume of wash buf fer Briefly rinse the microarray and then remove the buffer by aspiration Repeat for a total of three short washes in PBS T IMPORTANT Cover the 4 well plates with aluminum foil at all times during both the reaction and washing steps to minimize light exposure which could quench the fluorescence of your protein sample V 6 For research use only Briefly rinse the microarrays three times with ddH O Drying Place clean room wipes or paper towels on the bottom of a black micro scope slide box will hold several microarrays or prepare plastic conical 50 ml tubes each will hold one microarray Remove the HuProt microarray from the 4 well plate and tap the edge lightly on a paper towel to remove excess fluid Do not touch the active sur EN fl ep Not for use in diagnostic proced
18. es Submerge the coverslipped HuProt microarray in a compartment and carefully remove the cover slip using fine nosed tweezers Be careful not to scratch the active surface of the microarray alternatively float off the cover slip by immersing the microarray in a larger volume of wash buffer Shake gently for 5 min at room temperature and then remove the wash buffer by aspira tion Repeat for a total of three washes IMPORTANT Cover the 4 well plates with aluminum foil at all times to minimize light exposure Briefly rinse the microarrays three times with ddH O Remove the HuProt microarrays from the 4 well plates with tweezers Tap the edge of the microarray lightly on a clean room wipe to remove excess fluid Do not touch the active surface of the microarray Drying Place clean room wipes or paper towels on the bottom of a black microscope slide box will hold several microarrays or prepare plastic conical 50 ml tubes each will hold one microarray Remove the HuProt microarray from the 4 well plates and tap the edge light ly on a clean room wipe to remove excess fluid Do not touch the active surface of the microarray Carefully slot the microarrays into the microscope slide box the microarrays will be perpendicular to the wipes lining the box If you are using 50 ml tubes carefully slide a single microarray lengthwise into the tube To remove excess fluid spin the microscope slide box or the 50 ml tubes con tain
19. g HuProt microarrays with aluminum foil to minimize light exposure which can quench the fluorescence 1 7 Washing e Add 4 0 ml TBS T to each compartment of the 4 well plate cover with foil and incu bate with gentle shaking for 10 min at room temperature Remove the buffer from a corner of the 4 well plate by aspiration Repeat for a total of three washes Briefly rinse the slides three times with ddH O 1 8 Drying Place clean room wipes or paper towels on the bottom of a black microscope slide box will hold several microarrays or prepare plastic conical 50 ml tubes each will hold one microarray Remove the HuProt microarray from the 4 well plates and tap the edge lightly on a clean room wipe to remove excess fluid Do not touch the active surface of the mi croarray Carefully slot the microarrays into the microscope slide box the microar rays will be perpendicular to the paper towels lining the base of the box If you are using 50 ml tubes carefully slide a single microarray lengthwise into the tube To remove excess fluid spin the microscope slide box or the 50 ml tubes con taining the microarrays in a centrifuge at 800 rpm for 3 min spinning at higher speeds may break the microarray After centrifugation carefully remove the HuProt microarrays and discard the clean room wipes 1 9 Scanning e The HuProt microarrays can be scanned immediately highly preferred or stored at 20 C in a lightproof bo
20. h use only Not for use in diagnostic procedures IMP Page 8 Assay Add monoclonal antibody sample to HuProt microarrays Carefully use aspiration to remove the blocking buffer from a corner of the 4 well plates that contain the immersed human proteome microarrays Do not touch the microarray surface For low volume samples 100 ul to 300 ul carefully pipette the prepared primary monoclonal antibody onto the active surface of the blocked HuProt microarray barcode side up Do not let the pipette tip touch the microarray surface Cover the microarray with a cover slip to minimize evaporation and place in a humidification chamber ORTANT Use a sample of at least 100 ul For high volume samples up to 3 0 ml carefully add the sample to the active top surface of the microarray in the 4 well plate Be careful not to touch the surface of the microarray Incubate with gentle shaking on an orbital shaker at room temperature for 1 hr Washing Upon completing the assay the microarrays are washed in 4 well plates For low volume reactions carried out in a humidification chamber the cover slip must be removed prior to washing Immerse the covered microarray in a compartment of a 4 well plate containing 4 0 ml of wash buffer Using fine nosed tweezers carefully lift off the cover slip starting from the barcoded end of the microarray taking care to not touch or scratch the active surface of the microarray An alternative method
21. ides that are coated with 3D polymers that contain functional groups This allows the protein samples to be immobilized on the glass by covalent bonding Storage and Handling IMPORTANT New HuProt microarrays must be stored in an ultra cold and dry environment HuProt microarrays are shipped in closed plastic slide holders on dry ice or with gel coolant sheets Upon arrival microarrays should immediately be stored at 80 C To ensure the best performance from the HuProt microarray e Wear gloves at all times e Do not touch the active surface of the microarray the surface where the bar code label is attached with hands with pipette tips or with tweezers The active surface should face up at all times e Handle microarrays only along the edge near the barcode using tweezers Do not let the HuProt microarray dry out at any time during the assay e When conducting low volume assays be very careful when adding glass cov er slips to the active surface of the microarray used to minimize evaporation Likewise when the assay is completed be careful when removing the cover slips from the microarrays prior to the washing steps If the microarray surface is scratched proteins printed on the glass may be smudged or removed One alternative is to immerse the covered microarray in a large volume of wash buf fer and then allow the cover slip to float off EN fl ep Page 2 NEXTGEN PROTEOMICS CDI Laboratories User
22. ing the microarrays in a centrifuge at 800 rpm for 3 min spinning at higher speeds may break the microarray After centrifugation carefully remove the HuProt microarrays and discard the clean room wipes EN fl ep Page 14 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 15 TABLE OF CONTENTS ASSAYS RNA Binding 13 References Barry G et al 2013 The long non cod ing RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia associated alternative spli cing Molec Psychiatry Apr 30 doi 10 1038 mp 2013 45 ePub ahead of print Donnelly CJ et al 2013 RNA Toxicity from the ALS FTD C9O0RF72 Expansion is Miti gated by Antisense Intervention Neuron 80 2 415 28 Fan B et al 2013 A human microarray identifies that the heterogeneous nuclear ribonucleoprotein K hd RNP K recognizes the 5 terminal sequence of the hepatitis C virus RNA Mol Cell Proteomics 13 1 84 92 For research use only Not for use in diagnostic procedures II 7 Scanning and Storage e The HuProt microarrays can be scanned immediately highly preferred or stored at 20 C in a lightproof box IMPORTANT Microarrays must be scanned within 3 days after the assay is performed Appendix for Fluorescently labeled RNA binding Assay Recipes PBS T see General Appendix Recipe 2 Blocking solution 10 ug ml ssDNA herring sperm
23. ipe 1X e 137 mM NaCl e 2 7mM KCl 10 mM Na HPO 2 H O 1 8 mM KH PO e 10X PBS APPENDIX e For 1 liter of 10X PBS stock buffer dissolve the following in 800 ml distilled water 80 0 g NaCl Recipes 22 20g KCI Prot Literature Citations 23 14 4 g mM Na HPO 2 H O 24g KH PO e Adjust pH to 7 4 and add distilled water to 1 liter Sterilize by autoclaving or filtration Store at room temperature e PBS T e Add 1 0 ml Tween 20 to 100 ml 10X PBS solution Make up to 1 liter with distilled water Stir until homogenous Tween 20 is very viscous and may stick to pipette tips a 10 solution is easier to dispense than the undiluted form Blocking Solution Recipes NOTE Blocking conditions vary depending on the protocol used Please refer to each sec tion for the correct recipes a D For research use only Not for use in diagnostic procedures Page 22 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 23 TABLE OF CONTENTS APPENDIX HuProt Literature Citations 23 For research use only Not for use in diagnostic procedures General Appendix HuProt Human Proteome Microarray Literature Citations Tarrant MK et al 2012 Regulation of CK2 by phosphorylation and O GIcNAcyla tion revealed by semisynthesis Nat Chem Biol 8 3 262 9 Jeong JS et al 2012 Rapid identification of monospecific monoclonal antibodies using a human proteo
24. me microarray Mol Cell Proteomics 11 6 0111 016253 Huang Y et al 2012 Global tumor protein p53 p63 interactome making a case for cisplatin chemoresistance Cell Cycle 11 12 2367 79 Hu CJ et al 2012 Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays Mol Cell Proteomics 11 9 669 80 Barry G et al 2013 The long non coding RNA Gomatu is acutely regulated in re sponse to neuronal activation and involved in schizophrenia associated alternative splicing Molec Psychiatry Apr 30 doi 10 1038 mp 2013 45 ePub ahead of print Lee YI et al 2013 Protein microarray characterization of the S nitrosoproteome Mol Cell Proteomics 13 63 72 Chen Y et al 2013 Bcl2 associated Athanogene 3 Interactome Analysis Reveals a New Role in Modulating Proteasome Activity Mol Cell Proteomics 12 10 2804 19 Donnelly CJ et al 2013 RNA toxicity from the ALS FTD C9ORF72 expansion is mitigated by antisense intervention Neuron 80 2 415 28 Fan B et al 2013 A human proteome microarray identifies that the heteroge neous nuclear ribonucleoprotein K hnRNP K recognizes the 5 terminal sequence of the hepatitis C virus RNA Mol Cell Proteomics 13 1 84 92 Fan Q et al 2014 Identification of proteins that interact with alpha A crystallin using a human proteome microarray Mol Vis 20 117 124 Deng RP et al 2014 Global Identification of O GlcNAc Transferase OGT In teractors by
25. n 200 pl RNA binding buffer Assay Add Labeled RNA to HuProt microarrays Remove the HuProt microarrays from the 4 well plates and tap the edge lightly on a clean room wipe or paper towel to remove excess buffer Place the microarray in a humidification chamber to prevent evaporation Carefully pipette the prepared RNA sample onto the active surface of the blocked HuProt microarray Be careful not to let the pipette tip touch the active surface Cover the microarray with a glass cover slip to prevent evaporation Cover the humidification chamber with aluminum foil to minimize light ex posure that may quench the fluorescence Incubate with gentle shaking for 1 hr at room temperature np fl py Page 13 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 14 TABLE OF CONTENTS ASSAYS RNA Binding 13 For research use only Not for use in diagnostic procedures 1 5 I11 6 If you do not have a humidification chamber you can adapt an empty yel low tip box for this purpose Place wet clean room wipes or paper towels in the base of the yellow tip box and replace the empty tip rack on top of the towels Carefully place up to four coverslipped HuProt microarrays on top of the rack and then close the lid to create a humid environment IMPORTANT Use a sample volume of at least 100 ul for the assay Washes Add 4 0 ml PBS T to each compartment in the 4 well plat
26. ray only on the edges of the glass at the end where the bar code is attached Be careful not to touch the active surface of the microarray Block the HuProt microarrays The active surface of the HuProt microarray is the surface with the barcode Carefully use fine nosed tweezers to remove one microarray from the plastic slide holder that is resting on dry ice Immediately submerge the HuProt microarray with the active surface facing up in a 4 well plate containing 3 0 ml blocking buf fer Incubate with gentle shaking for 5 min Carefully pour off the blocking buffer or remove it by aspiration Add 3 0 ml fresh blocking buffer to the well of the plate and incubate as directed in the protocol Remove the blocking buffer from a corner of the 4 well plate via aspiration Use the microarrays immediately in the assay of choice see below IMPORTANT Blocking solution recipes vary by protocol please refer to the protocol of interest for the correct recipe EN fl py Page 3 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 4 TABLE OF CONTENTS SUMMARY 1 Overview Key Steps for HuProt Microarray Use 3 For research use only Not for use in diagnostic procedures Sample Preparation and Assay e High volume assays If a high volume of sample is available to test dilute it in 3 0 ml of buffer Add this to one compartment of a 4 well plate Immerse the HuProt micro
27. sing the HuProt Human Proteome Microarray V V 1 V 2 V 3 V 4 Fluorescently Labeled Protein Binding Assay Storage Store HuProt microarrays in closed plastic slide holders at 80 C or on a layer of dry ice right until the blocking step The active surface of the HuProt microarray is the surface with the barcode IMPORTANT It is critical to keep the HuProt microarrays ultra cold and ultra dry and to prevent liquid from condensing onto the microarray surface before use Blocking Add 3 0 ml of blocking solution 5 BSAin PBS T to each compartment of a 4 well plate Carefully use fine nosed tweezers to remove one microarray from the plastic slide holder resting on dry ice Immediately submerge the HuProt microarray active side up in a compartment containing block ing solution Incubate with gentle shaking for 5 min Carefully pour off the blocking buffer or remove it by aspiration Add 3 0 ml fresh blocking buffer to the well of the plate and incubate the microarray with gentle shaking for 1 5 2 hrs at room temperature Sample Preparation High volume assay Dilute 3 ug of the fluorescently labeled protein to be tested in 3 0 ml blocking solution Low volume assay If a limited amount of protein is available for testing dilute 200 ng of labeled protein in 200 ul of blocking solution Assay Add Labeled Protein to HuProt micorarrays High volume assay Add 3 0 ml of diluted labeled protein
28. t microarray active side up in a 4 well plate containing blocking buffer Incubate with gentle shaking for 5 min Carefully pour off the block ing buffer or remove it by aspiration Add 3 0 ml fresh blocking buffer to the well of the plate and incubate the microarray with gentle shaking for 1 5 2 hrs at room temperature Sample preparation The recommended serum dilution is 1 500 in blocking buffer Dilute the primary sample to a final volume of 3 0 ml in blocking buffer then vortex briefly and store on ice Assay Add Sample to HuProt Microarray Remove the blocking buffer out of a corner of the 4 well plates containing the immersed HuProt microarrays by aspiration Carefully pipette the prepared serum sample 3 0 ml onto the active sur face of the blocked HuProt microarray barcode facing up Be careful not to let the pipette tip touch the microarray surface Incubate with gentle shaking on an orbital shaker for 1 hr at room temperature eeeee ee ae eee ee aca am Page 10 NEXTGEN PROTEOMICS CDI Laboratories User Guide ILS HuProt Human Proteome Microarray v2 0 Washing Following incubation remove the buffer from the corner of the 4 well plate by aspiration Add 4 0 ml of TBS T to each well and rinse briefly Repeat for a total of three short washes Add 4 0 ml of TBS T and incubate 10 min at room temperature with gentle shaking Remove the buffer by aspiration from the corner of the 4 well plate
29. ures Pa ge 20 NEXTGEN PROTEOMICS CDI Laboratories User Guide face of the microarray Carefully slot the microarrays into the microscope slide box the microarrays will be perpendicular to the paper towels lining HuProt the box If you are using conical plastic 50 ml tubes carefully slide a single Human Proteome Microarray v2 0 microarray lengthwise into the tube To remove excess fluid spin the microscope slide box or the 50 ml tubes Page 21 containing the microarrays in a centrifuge at 800 rpm for 3 min spinning at higher speeds may break the microarray After centrifugation carefully remove the HuProt microarrays and discard the clean room wipe TABLE OF CONTENTS V 7 Scanning and Storage e The HuProt microarrays can be scanned immediately highly preferred or stored at 20 C in a lightproof box IMPORTANT Microarrays must be scanned within 3 days after the assay is performed ASSAYS Appendix for protein binding assay using fluorescently labeled protein probes Recipes PBS T see General Appendix Buffer Recipe Blocking solution 5 BSA in PBS T Protein Binding 19 References Huang Y etal 2012 Global tumor protein p53 p63 Interactome making a case for cisplatin chemoresistance Cell Cycle 11 12 2367 79 Chen Y et al 2013 Bcl2 associated Atha nogene 4 Interactive Analysis Reveals a New Role in Modulating Proteosome Activity Mol Cell Proteomics 12 10 2804 19 Fan Q et al 2014 Identifi
30. visiae purified and printed on glass slides in duplicate along with control proteins e H1 Histone H1 e GST10n glutathione S transferase at 10 ng ul e H2 A B Histone H2A and H2B mixture e GST50n GST at 50 ng pl e H3 Histone H3 e GST100n GST at 100 ng l e H4 Histone H4 all these histones are non specific e GST200n GST at 200 ng ul binding proteins used as positive controls for all Mouse anti biotin kinds of assays IgG488 594 Alexa Fluor 488 594 labeled IgG positive control and landmarks for fluorescent Rabbit anti biotin e Biotin BSA biotinylated BSA detection in 488 594 channels e BSA Bovine serum albumin negative control e 1gG555 647 Alexa Fluor 555 647 labeled IgG Buffer printing buffer only negative control positive control and landmarks for fluorescent Mouse IgM detection in 555 647 channels Summary continued on page 2 y CDI NEXTGEN PROTEOMICS e e ogee CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 2 TABLE OF CONTENTS SUMMARY 1 Storage and Handling 2 For research use only Not for use in diagnostic procedures Summary continued from page 1 These expressed recombinant proteins are N terminal GST and RGS His6 tagged and the quality of each microarray batch is determined by GST immunoblotting 98 of all proteins show GST signals significantly higher than negative controls For most applications we print the microarray on glass sl
31. x IMPORTANT Microarrays must be scanned within 3 days after after the assay is performed Appendix for Monoclonal Antibody Specificity Determination Recipes TBS T Buffer TBS pH 7 5 0 1 Tween 20 see General Appendix Buffers section Blocking Solution 5 BSA w v in TBS T Buffer Dissolve 5 g of Bovine Serum Al bumin lgG Free Protease Free in 60 ml of TBS T Complete to 100 ml with TBS T and filter before use Page 9 CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 10 TABLE OF CONTENTS ASSAYS Serum Profiling 10 For research use only Not for use in diagnostic procedures Assays Using the HuProt Human Proteome Microarray l1 1 II 2 I1 3 Serum Profiling Assay NOTE Typically a 1 500 dilution of serum in blocking buffer is used Storage Store HuProt microarrays in closed plastic slide holders at 80 C or on a layer of dry ice right until the blocking step The active surface of the HuProt microarray is the surface with the barcode IMPORTANT It is critical to keep the HuProt microarrays ultra cold and ultra dry prior to use Do not let liquid condense onto the microarray surface before use Blocking Add 3 0 ml of blocking solution 5 BSA TBS T to each compartment in the 4 well plates Carefully use fine nosed tweezers to remove one microarray from the plas tic slide holder that has been resting on dry ice Immediately submerge the HuPro
32. y use fine nosed tweezers to remove one microarray from the plastic slide holder that is resting on dry ice Immediately submerge the HuProt microarray active surface up in a compartment of the 4 well plate containing blocking buffer Incubate with gentle shaking for 5 min Carefully pour off the blocking buffer or remove it by aspiration Add 3 0 ml fresh blocking buffer to the well of the plate and incubate the microarray at room tem perature for 1 5 2 hrs with gentle shaking Sample Preparation Prepare the primary monoclonal antibody for testing Low volume reaction If the primary antibody to test is available in a limited amount dilute the primary antibody to a concentration of 400 ng ml in 300 ul blocking buffer Store on ice High volume reaction If the amount of primary antibody to be tested is not limiting dilute the primary antibody to a concentration of 400 ng ml in 3 0 ml blocking buffer Store on ice If the antibody source is in supernatant form Make a 1 12 dilution of the supernatant in blocking buffer diluted to a final volume of 300 ul Store on ice NOTE This dilution assumes that the antibody concentration is 0 005 mg ml of supernatant Please base your dilution on the actual antibody concentration of your sample EN fl ep Page 7 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 8 TABLE OF CONTENTS ASSAYS Antibody Specificity 7 For researc
33. you can adapt an empty yellow tip box for this purpose Place wet clean room wipes or pa per towels at the base of the yellow tip box and replace the empty tip rack above the towels Carefully place up to four coverslipped HuProt microarrays on the rack and then close the lid Sample Preparation Low volume assay Dilute the fluorescently labeled DNA sample to 40 nM in 200 yl of DNA hybridization buffer containing poly dA dT Assay Add Labeled DNA to the HuProt microarrays After blocking the cover slip must be removed Immerse the blocked mi croarray in 4 0 ml of pre chilled wash buffer and carefully remove the cover slip using fine nosed tweezers being careful not to scratch the active sur face of the microarray alternatively float the cover slip off by immersing the microarray in a larger volume of wash buffer Drain off residual buffer by tapping the microarray sideways on clean room wipes EN fl ep Page 16 NEXTGEN PROTEOMICS CDI Laboratories User Guide HuProt Human Proteome Microarray v2 0 Page 17 TABLE OF CONTENTS ASSAYS DNA Binding 16 For research use only Not for use in diagnostic procedures IV 5 IV 6 Place the HuProt microarrays back in the humidification chamber Im mediately pipette the fluorescently labeled DNA sample 200 ul onto the active surface of the HuProt microarray Do not let the pipette tip touch the microarray surface Cover the microarray with a glass

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User Guide HuProt™ Human Proteome Microarray v2.0 User