Quantibody Human MMP Array 1 RayBiotech, Inc.
Contents
1. Please make sure to cover the incubation chamber tightly to prevent evaporation Quantibody Human MMP Array 1 IV Protocol A Completely air dry the glass chip 1 Take out the glass chip from the box and let it equilibrate to room temperature inside the sealed plastic bag for 20 30 minutes Remove slide from the plastic bag peel off the cover film and let it air dry at room temperature for another 1 2 hours Note Incomplete drying of slides before use may cause the formation of comet tails B Prepare Cytokine Standard Dilutions Note There is only one vial of standard provided in the two slide kit which is enough for making two standard curves Reconstitute the lyophilized standard within one hour of usage If you must use the standard for two different days store only the 8111 dilution at 80 C Prepare serial dilution of cytokine standards 100ul 100ul 100ul 100ul 100ul 100ul A 303 OS O n n Add 500u1 Sample Diluent 2001 200ul 200 200u 2000 200g 100 Vial Labels Std1 Std2 Std3 Std4 Std5 Std6 Std7 CNTRL 2 Reconstitute the Cytokine Standard Mix lyophilized by adding 5001 Sample Diluent to the tube For best recovery always quick spin vial prior to opening Dissolve the powder thoroughly by a gentle mix Labeled the tube as Std1 Quantibody Human MMP Array 1 8 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200ul Sample Diluent to each of the tubes 4 Pipette 1004 Stdl2. Fluor dye cy3 equivalent Biotin Streptavidin complex Detect antibody Cytokine Capture antibody Glass Slide Support Quantibody Human MMP Array 1 1 TABLE OF CONTENTS 1 3 How IU W OLKS 5252229245 5 Materials 2 22 6 Additional Materials 6 General 7 A Preparation of 7 B Handling Glass 7 ieee 7 8 A Complete Air Dry Glass Chip 8 B Prepare Cytokine Standard Dilutions 8 C Blocking and Incubation 9 D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 Fluorescence 11 G D ta X dlys185422120 99 05 822 U1Ud3 Edad Ruou Pado du dd 12 V Cytokine Array Map amp Standard Curves 13 VI 8 Point Standards sies exem eo diae a l4 VIIL System Recovery 15 VIII Quantibody Q Analyzer ep Cobrri ERE 16 IX Troubleshooting 17 X Select Quantibody Publications 18 XI Experi
3. based array Custom Arrays Choose from over 400 cytokine pool Any kind Any number Order slide only or full service in house Check our website regularly for updated Quantibody products Quantibody Human MMP Array 1 20 Note Quantibody is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for three months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price This product is for research use only 2010 RayBiotech Inc Quantibody Human MMP Array 1 21
4. into tube Std2 and mix gently Perform 5 more serial dilutions by adding 100ul Std2 to tube Std3 and so on 5 Add 1004 Sample Diluent to another tube labeled as CNTRL Do not add standard cytokines or samples to the CNTRL tube which will be used as negative control For best results include a set of standards in each slide Note Since the starting concentration of each cytokine is different the serial concentrations from 5141 to Std7 for each cytokine are varied which can be found in section VI C Blocking and Incubation 6 Add 100ul Sample Diluent into each well and incubate at room temperature for 30 min to block slides 7 Decant buffer from each well Add 10011 standard cytokines or samples to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable for higher signals Note We recommend using 50 to 100 ul of original or diluted serum plasma conditioned media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Cover the incubation chamber with adhesive film during incubation if less than 70 ul of sample or reagent is used Note This step may be done overnight at 4c for best results 8 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 of 1 Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer I with H O Quantibody Human MMP Array 1 9
5. reading yet remains unsaturated Note In case the signal intensity for different cytokine varies greatly in the same array we recommend using multiple scans with a higher PMT for low signal cytokines and a low PMT for high signal cytokines Quantibody Human MMP Array 1 11 G Data Analysis 19 Data extraction can be done with most of the microarray analysis software GenePix ScanArray Express Array Vision or MicroVigene For quantitative data analysis our Quantibody Q Analyzer software is available It gives visual output as well as digital values More information can be found in section VIII Experiments 9 Image scan laser scanner Data extraction 455 433 443 442 121 122 132 119 2 1 3 2 89 88 90 91 GenePix etc ee 55 54 57 56 188 178 189 190 Data computation Q Analyzer Final Result pg ml Quantibody Human MMP Array 1 12 V Cytokine Array Map amp Standard Curves POS1 POS2 1 2 3 8 9 10 13 1 2 4 1 100000 10000 1000 pi NEL 10 1 100 1000 10000 100000 1000000 Concentration pg ml 10000 MMP 2 ee cue 1000 100 100 1000 10000 100000 Concentration pg ml 100000 MMP 3 10000 S
6. software can automatically mark and remove the outlier spots for more accurate data analysis e Normalization The program allows for intra and inter slide normalization for large number of samples e Two Positive Controls The program takes the two positive controls in each array for normalization e Two Analytical Algorithms Users can choose either linear regression or log log algorithms to meet their analytical needs e Two Data Outputs standard curves and digital concentration e User Intervention The program allows for user manual handling of those outliers and other analytical data e Lower and Upper Limits Determination The program automatically marks out the values below or above the detection range e Standard Deviation The program outputs the standard deviations of the quadruplicate spots for data accuracy e Analytical Tips Q Analyzer analysis tips are included in the program Quantibody Human MMP Array 1 16 IX Troubleshooting guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or improper dilution Check pipettes and ensure correct preparation Short incubation time Weak Signal Ensure sufficient incubation time and change sample incubation step to overnight Too low protein concentration in sample Don t make too low dilution or concentrate sample Improper storage of kit Store kit as suggested tempera
7. 1 QAH INF 1 QAH INF 2 QAH INF 3 QAH CYT 1 QAH CYT 2 QAH MMP 1 QAH ISO 1 QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 QAH ADI 1 QAH ADI 2 QAH CHE 1 QAH GF 1 QAH REC 1 QAH CAA 1000 QAH CAA 2000 QAH CAA 3000 QAH CAA 4000 QAH CAA 5000 QAH TH 17 Mouse QAM CYT 1 QAM CYT 2 3 QAM CYT 4 QAM CYT 5 QAM CYT 6 QAM INF 1 QAM INT 1 QAM INT 2 QAM INT 1000 QAM CAA 1000 QAM CYT Q2000 QAM CAA 2000 QAM TH 17 Rat QAR CYT 1 QAR CYT 2 QAR CYT 3 QAR INF 1 Porcine QAP CYT 1 Function based arrays THI TH2 TH17 Arrays QAH TH 1 QAH TH 17 QAM TH 17 Inflammation Arrays QAH INF 1 QAH INF 2 QAH INF 3 QAM INF 1 QAR INF 1 Angiogenesis Arrays QAH ANG 1 2 QAH ANG 3 QAH ANG 1000 MMP Array QAH MMP 1 Immunoglobin Isotype Array QAH ISO 1 Cytokine Number based arrays 240 cytokines QAH CAA 5000 200 cytokines QAH CAA 4000 160 cytokines QAH CA A 3000 120 cytokines QAH CAA 2000 QAM CAA 2000 80 cytokines QAH CAA 1000 QAM CAA 1000 60 cytokines QAH ANG 1000 QAM CYT Q2000 40 cytokines QAH INF 3 1 QAH GF 1 QAH REC 1 QAM INF 1 QAM CYT 4 QAM CYT 5 QAM CYT 6 QAH CY T 4 QAH CYT 5 20 30 cytokines QAH ANG 2 QAH ANG 3 QAM INT 1000 QAR CYT 3 20 cytokines QAH CYT 1 QAM CYT 1 QAM CYT 2 QAM CYT 3 QAM INT 1 10 or less QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1 QAH ADI 1 QAM INT 2 QAR CYT 1 2 QAR INF 1 QAH ISO 1 QAP CYT 1 Purpose
8. Quantibody Human MMP Array 1 Quantitative measurement of 10 human Matrix Metalloproteinase related proteins Patent Pending Technology User Manual Version July 2010 Cat t QAH MMP 1 RayBiotech Inc We Provide You With Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com Cytokine Detected MMP 1 MMP 2 MMP 3 MMP 8 MMP 9 MMP 10 10 13 TIMP 1 TIMP 2 TIMP 4 One standard glass slide is spotted with 16 wells of Format identical cytokine antibody arrays Each antibody is arrayed in quadruplicate Detection Method Fluorescence with laser scanner Cy3 equivalent dye Sample Volume 50 100 ul per array Reproducibility CV lt 20 Assay duration 6 hrs 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 See Section V For Array Map
9. Recovery Rate pg ml pg ml Ag pg ml y 18x Human Serum 1965 1694 156 7896 18x Conditioned Media 1965 2505 11 12796 Spiked Ag Observed Ag Endogenous pg ml pg ml Aa pami Recovery Rate 18x Human Serum 7186 14542 4057 14696 18x Conditioned Media 7186 5911 312 78 Spiked Ag Observed Ag Endogenous Salt Ag po mi Recovery Rate 18x Human Serum 126892 181388 44214 108 18x Conditioned Media 126892 165323 1152 129 Spiked Ag Observed Ag Endogenous Ag po mi Recovery Rate 18x Human Serum 4066 2354 53 57 18x Conditioned Media 4066 3794 117 90 Spiked Ag Observed Ag Endogenous TIMP 1 pg ml pg ml Ag pg ml Recovery Rate 18x Human Serum 2632 13758 11729 77 18x Conditioned Media 2632 2750 97 101 Spiked Ag Observed Ag Endogenous TIMP 2 pg ml pg ml Ag pg ml Recovery Hale 18x Human Serum 1866 13724 12075 88 18x Conditioned Media 1866 1287 13 68 Spiked Ag Observed Ag Endogenous TIMP 4 pg ml pg ml Ag pg ml Recovery Rate 18x Human Serum 8516 6387 1228 61 18x Conditioned Media 8516 4868 205 55 Quantibody Human MMP Array 1 15 VIII Quantibody Q Analyzer Quantibody Q Analyzer is an array specific Excel based program However it is not a simple calculation macro as it contains sophisticated data analysis Key features e Simplicity Easy to operate and requires no professional training With simple copy and paste process the cytokine concentration is determined e Outlier Marking amp Removing The
10. a H 1000 Pad 100 G 10 1 10 100 1000 10000 100000 Concentration pg ml MMP 8 100000 10000 H 1000 100 B 10 1 10 100 1000 10000 100000 Concentration pg ml MMP 9 10000 1000 ae 189 e v 10 1 10 100 1000 10000 100000 Concentration pg ml Quantibody Human MMP Array 1 10 100000 10000 Pd Signal BKG 1000 100 eee 10 4 10 100 1000 10000 Concentration pg ml 100000 MMP 13 10000 use e g 1000 100 P ud Signal md d 10 100 1000 10000 100000 Concentration pg ml TIMP 1 100000 10000 ee 1000 300 lt 2 10 10 100 1000 10000 100000 Concentration pg ml TIMP 2 100000 10000 1000 ge P 100 10 10 100 1000 10000 100000 Concentration pg ml TIMP 4 10000 m5 1000 Ed 100 Signal BKG Que 10 10 100 1000 10000 Concentration pg ml 100000 VI 8 Point Standards After reconstitution of the lyophilized cytokine standard mix the 8 point cytokine concentration used for generating the standard curve of a given antigen is listed below The detection sensitivity of each protein in one experiment is user dependent Try our array specific Quantibo
11. de chips can be nested into a tray which matches a standard microplate and allows for automated robotic high throughput process of 64 arrays simultaneously For cytokine quantification the array specific cytokine standards whose concentration has been predetermined are provided to generate a standard curve for each cytokine In a real experiment standard cytokines and samples will be assayed in each array simultaneously through a sandwich ELISA procedure By comparing signals from unknown samples to the standard curve the cytokine concentration in the samples will be determined Quantibody array kits have been confirmed to have similar detection sensitivity as traditional ELISA Our current high density Quantibody kits allow scientists to quantitatively determine the concentration of 160 human or 120 mouse cytokines in a single experiment This is not only one of the most efficient products on the market for cytokine quantification but makes it more affordable for quantification of large number of proteins Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool for drug and biomarker discovery Quantibody Human MMP Array 1 4 How It Works Array support YYYYY a Incubation of Sample vYYYY With arrayed antibody 1 2 br Samples Supports Cocktail of Biotin Ab KK S Incubation with YYY Biotinylated Ab 1 2 hr Labeled gaa a l streptavidin T K Incubation with Y Cy3 equival
12. dy Q Analyzer to see your Limit of Detection LOD Section VIIT Serial standard concentration pg ml pg ml Cntrl Std7 Std6 Std5 Std4 Std3 Std2 Std MMP 1 0 137 412 1 235 3 704 11 111 33 333 100 000 MMP 2 0 69 206 617 1 852 5 556 16 667 50 000 MMP 3 0 41 123 370 1 111 3 333 10 000 30 000 8 0 27 82 247 741 2 222 6 667 20 000 MMP 9 0 41 123 370 1 111 3 333 10 000 30 000 MMP 10 0 7 21 62 185 556 1 667 5 000 13 0 27 82 247 741 2 222 6 667 20 000 TIMP 1 0 27 82 247 741 2 222 6 667 20 000 2 0 P 82 247 741 2 222 6 667 20 000 TIMP 4 0 69 206 617 1 852 5 556 16 667 50 000 Quantibody Human MMP Array 1 VII System Recovery The antibody pairs used in the kit have been tested to recognize their specific antigen The spiking recovery rate of the cytokines by the kit in 18x diluted human serum and human conditioned media is listed in the following table Spiked Ag Observed Ag Endogenous pg ml pg ml Aa og ml Recovery Rate 18x Human Serum 8108 5814 69 7196 18x Conditioned Media 8108 9308 5 11596 Spiked Ag Observed Ag Endogenous Recovery Rate pg ml pg ml Ag pg ml 18x Human Serum 7479 5141 63 68 18x Conditioned Media 7479 8602 8 11596 Spiked Ag Observed Ag Endogenous Saif Ag po mi Recovery Rate 18x Human Serum 3573 4056 1534 71 18x Conditioned Media 3573 2016 0 5696 Spiked Ag Observed Ag Endogenous
13. e Optional for Cell and Tissue Lysates Put the glass chip with frame into a box with 1x Wash Buffer I cover the whole glass slide and frame with Wash Buffer I and wash at room temperature with gentle shaking for 20 min e Decant the Wash Buffer I from each well wash 2 times 5 min each with 150 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer II with H5O Note Incomplete removal of the wash buffer in each wash step may cause dark spots Background signal is higher than that of the spot D Incubation with detection antibody cocktail and wash 9 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly 10 Add 80 ul of the detection antibody cocktail to each well Incubate at room temperature for 1 2 hour Longer incubation time is preferable for higher signals and backgrounds 11 Decant the samples from each well and wash 5 times with 150 ul of Ix Wash Buffer I and then 2 times with 150 of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step E Incubation with Cy3 equivalent dye Streptavidin and wash 12 After briefly spinning down add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently 13 Add 80 ul of Cy3 equivalent dye conjugated streptavidin to each well Cover the device with alumi
14. ent dye hr Labeled streptavidin Detection of signals Data analysis and graph Quantibody Human MMP Array 1 II Materials Provided Upon receipt all components of the Quantibody Array kit should be stored at 20 C At 20 C the kit will retain complete activity for up to 6 months Once thawed the glass chip cytokine standard mix detection antibody cocktail and Cy3 equivalent dye conjugated Streptavidin should be kept at 20 and all other components may be stored at 4 The entire kit should be used within 6 months of purchase Components Item Description 1 Slide kit 2 Slide kit Quantibody Array Glass Chip Sample Diluent 20X Wash Buffer I 20X Wash Buffer II Lyophilized cytokine standard mix Detection antibody cocktail Cy3 equivalent dye conjugated Streptavidin Slide Washer Dryer Adhesive device sealer Manual mm 2 3 4 5 6 7 8 9 See Section VI for detailed cytokine concentrations after reconstitution Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5ml Polypropylene microcentrifuge tubes Quantibody Human MMP Array 1 6 General Considerations A Preparation of Samples Use serum free conditioned media if possible If serum containing conditioned media is required it is highly recommended that complete medium be
15. f human autoantibodies that may be used for the treatment of prion diseases Alzheimer s and Dementia 2009 4 4 T484 T484 Van Rossum et al Granulocytosis and thrombocytosis in renal cell carcinoma a pro inflammatory cytokine response originating in the tumour Neth J Med 2009 67 5 191 4 10 Zhai et al Coordinated Changes in mRNA Turnover Translation and RNA Processing Bodies in Bronchial Epithelial Cells following Inflammatory Stimulation Molecular and Cellular Biology 2008 28 24 7414 7426 11 Gao et al A Chinese herbal decoction Danggui Buxue Tang activates extracellular signal regulated kinase in cultured T lymphocytes FEBS Letters 2007 581 26 5087 5093 This reference validates mulitplex ELISA results for several analytes with standard ELISA test results 12 Piganelli et al Autoreactive T cell responses new technology in pursuit of an old nemesis Editorial Review Pediatric Diabetes 2007 8 249 251 Quantibody Human MMP Array 1 18 XI Experiment Record Form Date File Name Laser Power PMT Well No Sample Name Dilution factor 1 CNTRL 2 Std7 3 Std6 4 Std5 5 Std4 6 Std3 7 Std2 8 Stdl 9 10 11 12 13 14 15 16 Quantibody Human MMP Array 1 19 B B Le Ed E 4L dhe Ihe The TJ XII How to Choose Quantibody Products Species based arrays Human QAH TH
16. mental Record 19 XII How to Choose Quantibody Products 2225 apps 20 Quantibody Human MMP Array 1 2 I Introduction Cytokines play an important role in innate immunity apoptosis angiogenesis cell growth and differentiation They are involved in interactions between different cell types cellular responses to environmental conditions and maintenance of homeostasis In addition cytokines are also involved in most disease processes including cancer and cardiac diseases The traditional method for cytokine detection and quantification 1s through the use of an enzyme linked immunosorbent array ELISA In this method target protein is first immobilized to a solid support The immobilized protein is then complexed with an antibody that is linked to an enzyme Detection of the enzyme complex can then be visualized through the use of a substrate that produces a detectable signal While the traditional method works well for a single protein the overall procedure is time consuming and requires a lot of sample With little sample to work with conservation of precious small quantities becomes a risky task Take the advantage of advancement in microarray technology over the last decade more and more choices are available to the scientist today A long standing leader in the field Raybiotech has pioneered the development of cytokine antibody arrays which has now been widely applied in the research communit
17. num foil to avoid exposure to light or incubate in dark room Incubate at room temperature for 1 hour Quantibody Human MMP Array 1 10 14 Decant the samples from each well and wash 5 times with 150 ul of Ix Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each wash step F Fluorescence Detection 15 Disassemble the device by pushing clips outward from the slide side Carefully remove the slide from the gasket Be careful not to touch the surface of the array side 16 Place the slide in the slide Washer Dryer a 4 slide holder centrifuge tube add enough 1x Wash Buffer I about 30 ml to cover the whole slide and then gently shake at room temperature for 15 minutes Decant Wash Buffer I Wash with 1x Wash Buffer II about 30 ml with gentle and gently shake at room temperature for 5 minutes 17 Remove water droplets completely by one of the following ways e Put the glass chip into the Slide Washer Dryer and dry the glass chip by centrifuge at 1 000 rpm for 3 minutes without cap e Or dry the glass chip by a compressed stream e Or gently apply suction with a pipette to remove water droplets Do not touch the array only the sides 18 Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength such as Axon GenePix Make sure that the signal from the well containing the highest standard concentration Std1 receives the highest possible
18. ord Blood Mononuclear Cells in Response to Diabetes associated Autoantigens Scandinavian Journal of Immunology 2009 70 2 149 158 Willingham SB et al NLRP3 NALP3 Cryopyrin facilitates in vivo caspase 1 activation necrosis and HMGBI release via inflammasome dependent and independent pathways J Immunol 2009 183 3 2008 15 El Karim et al Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Journal of Endodontics 2009 35 6 829 833 Souquiere S et al T Cell tropism of simian T cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills Mandrillus sphinx J Med Primatol 2009 38 4 279 89 Sharma et al Induction of multiple pro inflammatory cytokines by respiratory viruses and reversal by standardized Echinacea a potent antiviral herbal extract Antiviral Research 2009 83 2 165 170 Altamirano Dimas et al Echinacea and anti inflammatory cytokine responses Results of a gene and protein array analysis Pharmacuetical Biology 2009 47 6 500 508 Cheung et al Cordysinocan a polysaccharide isolated from cultured Cordyceps activates immune responses in cultured T lymphocytes and macrophages Signaling cascade and induction of cytokines Journal of Ethonopharmacology 2009 124 1 61 68 Du et al P2 380 Identification and characterization o
19. ture Don t freeze thaw the slide Bubble formed during incubation Avoid bubble formation during incubation Arrays are not completed covered by Uneven signal reagent Completely cover arrays with solution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overflowing wash buffer Comet tail formation Air dry the slide for at least 1 hour before usage Inadequate standard reconstitution or Improper dilution Poor standard Reconstitute the lyophilized standard well at the room temperature before making serial dilutions Check pipettes and ensure proper curve serial dilutions Inadequate detection Increase laser power that the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Use freeze thawed cytokine standards Always use new cytokine standard vial for new set of experiment Discard any leftover Overexposure Lower the laser power Dark spots Completely remove wash buffer in each wash step High Insufficient wash Increase wash time and use more wash background buffer Dust Work in clean environment Slide is allowed to dry out Don t dry out slides during experiment Quantibody Human MMP Array 1 17 Select Quantibody Publications Stechova et al Influence of Maternal Hyperglycaemia on C
20. used as a control since many types of sera contains cytokines We recommend the following parameters for your samples 50 to 100 ul of original or diluted serum plasma cell culture media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates If you experience high background or the readings exceed the detection range further dilution of your sample is recommended Handling glass chips Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only Handle all buffers and slides with latex free gloves Handle glass chip in clean environment Because there is no barcode on the slide transcribe the slide serial number from the slide bag to the back of the slide with a permanent marker before discarding the slide bag Once the slide is disassembled you might not have enough info to distinguish one slide from the other C Incubation Completely cover array area with sample or buffer during incubation Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or 70 of sample or reagent is used Several incubation steps such as step 6 blocking step 7 sample incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may be done overnight at 4
21. y with hundreds of peer reviewed publications such as in Cell and Nature Quantibody array our quantitative array platform uses the multiplexed sandwich ELISA based technology and enables researchers to accurately determine the concentration of multiple cytokines simultaneously It combines the advantages of the high detection sensitivity specificity of ELISA and the high throughput of the arrays Like a traditional sandwich based ELISA it uses a pair of cytokine specific antibodies for detection A capture antibody is first bound to the glass surface After incubation with the sample the target cytokine is trapped on the solid surface A second biotin labeled detection antibody is then added which can recognize a different isotope of the target cytokine The cytokine antibody biotin complex can then be visualized through the addition of the streptavidin labeled Cy3 equivalent dye using a laser scanner Unlike the traditional ELISA Quantibody products use array format By arraying multiple cytokine Quantibody Human MMP Array 1 3 specific capture antibodies onto a glass support multiplex detection of cytokines in one experiment is made possible In detail one standard glass slide is spotted with 16 wells of identical cytokine antibody arrays Each antibody together with the positive controls is arrayed in quadruplicate The slide comes with a 16 well removable gasket which allows for the process of 16 samples in one slide Four sli
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