USER MANUAL SUPPLEMENT:
Contents
1. USER MANUAL SUPPLEMENT Assay Solution for Invitrogen AKT pS473 Antibody Pair Invitrogen Catalog Number CHO0115 Powered By PTIMISER MICROPLATES An ELISA for AKT pS473 utilizing reagents from LIFE Technologies Invitrogen Catalog Number CHOO115 has been successfully transferred from a conventional 96 well ELISA plate format to the Optimiser microplate platform to achieve the following key performance benefits Sample Volume 5ul Assay time Total assay time 2 hours 3 5 hour savings Assay reagents 80 saving on antibody use 5 assays for the cost of 1 Sensitivity Range 4x improvement 0 39 100 Units ml e Potential to increase sensitivity to 0 02 U ml e Potential to achieve gt 2 log dynamic range 0 39 285 U ml Intended Use This User Manual Supplement is intended to be used in conjunction with Invitrogen s Technical Data Sheet for the AKT pS473 Antibody Pair Catalog Number CHO0115 employed in this Optimiser based ELISA procedure Please refer to the vendor s instructions for material storage preparation and concentration information The procedure described in this User Manual Supplement is intended as a starting reference for the investigator using the vendor s assay reagents with the Optimiser microplate system Siloam has optimized this procedure using OptiBlock as blocking solution as diluent for the anti rabbit IgG HRP conjugate and as the media to reconstitute the2. a ___ Observe the plate periodically during this incubation When the substrate solution has drained from all wells first remove the plate from the holder and then remove the pad from the plate b ___ Wipe the bottom of the plate thoroughly with a KimWipe or similar laboratory tissue ___ Read the plate using an excitation wavelength of 528 20 nm and an emission wavelength of 590 35 nm ___ This space is provided as a simple way of documenting the completion of each step Page 8 of 13 Page 9 of 13 Calculations 1 Calculate the mean background signal RFU 2 Subtract the mean background signal from the individual standard and sample values Calculate the mean background adjusted signal for each standard and sample 4 Prepare a standard curve by plotting the concentration of the standard on the x axis and the background adjusted signal on the y axis using a 4 parameter curve fit 5 Interpolate the sample concentrations from the standard curve Calculate the final concentration after applying the sample dilution factor if applicable w Typical Data The standard curve illustrated below was generated using the method reagents and equipment specified in this procedure 5500 Optimiser Based ELISA for AKT pS473 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 Bkg Adjusted RFU 0 20 40 60 80 100 AKT pS473 U mL Page 10 of 13 Page 11 of 13 Additional technical assis
3. Antibody Capture Ab AKI Paal Bete enon Antibody gerection Invitrogen CH00115 Per TDS Anti Rabbit IgG HRP Conjugate HRP conjugate Reagent Diluent Diluent R amp D Systems DY995 Per TDS Optimiser Materials Material Product Number Storage Optimiser plate with holder 2 OPH 10 Room temp OptiMax buffer reagent pack with substrate OMR 10 G 2 8 C Other Material amp Equipment Materials Equipment Polypropylene centrifuge tubes 1 5 2 mL Fluorescence plate reader snap cap Pipet tips Vortex mixer KimWipes Single channel pipettor s Test tube rack Multichannel pipettor Reagent reservoirs v bottom 96 well polypropylene v bottom plate Refer to the respective Technical Data Sheets TDS for storage concentration and other relevant information Optimiser plates and corresponding OptiMax buffer reagents are also available in 2 plate and 50 plate configuration Page 3 of 13 REAGENT PREPARATION 1 Working concentrations of all materials should be prepared before beginning the procedure 2 Capture antibody working solution a Refer to the vendor s TDS for the protein concentration of the stock capture antibody solution b Prepare the capture antibody working solution by diluting the stock capture antibody 1 62 5 in OptiBind G 3 Lyophilized Standard a Reconstitute the lyophilized protein standard with OptiBlock Refer
4. protein standard R amp D Systems Reagent Diluent has been used as diluent for the standards and detection antibody Siloam has not evaluated this procedure for its applicability for analysis of tissue culture supernatants serum or plasma samples Use of this assay for the analysis of tissue culture supernatant serum plasma or other sample types may require further optimization of some assay parameters by the investigator for example standard curve and sample diluents to achieve desired results It is expected that investigators following this assay procedure are familiar with the Optimiser microplate system If you have not used the Optimiser microplate before please order the Evaluation kit Catalog OPV IL6 which provides a comprehensive overview to the Optimiser microplate system The Evaluation Kit guides the user through correct pipetting procedures for Optimiser microplates and contains all necessary materials and instructions for completing an illustrative human IL 6 assay Investigators are strongly urged to familiarize themselves with the Optimiser microplate system before completing the procedure described in this document Please contact Siloam s tech support techsupport siloambio com for any questions regarding this procedure FOR RESEARCH USE ONLY Not for Use in Diagnostic Procedures MATERIALS Assay Reagents Material Function Vendor Catalogue Storage Number AKT Coating
5. to the vendor s TDS for further directions the concentration of the reconstituted standard and storage conditions 4 Standard Curve a Prepare standard 1 by diluting the reconstituted standard to 100 Units mL in Reagent Diluent b Dispense 200 uL of standard 1 to well A1 of the polypropylene 96 well v bottom plate c Dispense 100 uL of Reagent Diluent to each of the 7 wells immediately below well A1 d Prepare serial two fold dilutions of the standard by successive 100 uL transfers through well G1 Change pipet tips after each transfer and mix the well contents 8 10 times by gently aspirating and dispensing the well contents Do not transfer standard to well H1 Well H1 will serve as the assay blank 0 pg mL 1 1 A 200 uL Std 1 A Std 1 B 100 uL OB B Std 2 C 100 uL OB c Std 3 D 100 uL OB gt gt D Std 4 E 100 uL OB E Std 5 F 100 uL OB F Std 6 G 100 uL OB G Std 7 H tO ule H Blank 5 Detection antibody a Prepare the detection antibody working solution by diluting the stock detection antibody material 1 250 in Reagent Diluent 6 Anti Rabbit IgG HRP Conjugate a Prepare the anti rabbit IgG HRP conjugate working solution by diluting the stock material 1 600 in OptiBlock 7 OptiGlow working solution a Prepare the OptiGlow substrate working solution by combining OptiGlow A OptiGlow B and OptiGlow C in proportions of 50 50 1 par
6. astic covered surface facing the holder c Position the pad and plate on the holder surface and push down firmly until the plate snaps into position 2 Reverse pipetting Introduction of bubbles to the Optimiser wells will compromise method performance by occluding the microchannel To avoid introducing bubbles always use the reverse pipetting technique when delivering materials to an Optimiser well a Beginning with the pipettor s operating button in the ready position depress the operating button to the second stop See figure in step c below b Immerse the pipet tip in the liquid to be transferred Aspirate the liquid by releasing the operating button returning it to the ready position c Dispense the liquid to the Optimiser well by depressing the operating button to the first stop Ensure that the pipet tip is touching the well surface as the liquid is dispensed Pipetting Step Ready Position 1 2 3 4 First Stop t Second Stop 3 Transferring reagents standards and samples to the Optimiser plate Due to the short incubation times it is critical that antibodies standards samples SAv HRP and substrate are transferred from their source to the Optimiser wells quickly lt 1 minute but accurately To accomplish this a First dispense the materials to a polypropylene 96 well v bottom plate b Then using a multichannel pipettor transfer the materials from the polypropylene v bottom plate to t
7. he Optimiser wells as illustrated in the figure on the next page Optimiser Washes Optimiser based ELISAs use a unique flush step rather than the traditional and laborious wash step used in conventional ELISAs To flush the user simply dispenses OptiWash into the Optimiser well The wash buffer flushes the used reagent sample from the microchannel into an absorbent pad beneath the plate The Optimiser flush is equally effective as the traditional washes Page 6 of 13 1 2 3 5 6 7 8 9 10 11 12 A Std 1 Samp Samp i 9 B Std 2 Samp Samp 2 10 C Std 3 Samp Samp 3 11 D Std 4 Samp Samp 4 12 E Std 5 Samp Samp 5 13 F Std 6 Samp Samp 6 14 G Std 7 Samp Samp 7 15 H Blank Samp Samp 8 16 M Polypropylene v bottom plate containing diluted standards samples and blank 5 uL of standard sample and blank are transferred from individual wells of polypropylene v bottom plate to duplicate cells of Optimiser 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Sample 1 Sample 9 B Std 2 Sample 2 Sample 10 C Std 3 Sample 3 Sample 11 D Std 4 Sample 4 Sample 12 Shaded cells not used in this assay E Std 5 Sample 5 Sample 13 F Std 6 Sample 6 Sample 14 G Std 7 Sample 7 Sample 15 H Blank Sample 8 Sample 16 0 pg mL M Optimiser plate to which standa
8. rds samples and blank will be dispensed Page 7 of 13 PROCEDURE 2 10 11 12 13 ___ Assemble the Optimiser plate pad and holder as described earlier ___ Dispense 5 uL of the working capture antibody solution to the appropriate number of wells in the Optimiser plate Incubate 10 minutes at room temperature RT ___ Following the incubation dispense 5 uL OptiWash to each well Incubate 10 minutes at RT ___ Following the wash step dispense 5 uL OptiBlock to each well Incubate 10 minutes at RT ___ Following the block step dispense 5 uL standard or sample to each well Incubate 20 minutes at RT ___ Following the sample incubation dispense 5 uL OptiWash to each well Incubate 10 minutes at RT ___ Following the wash step dispense 5 uL of the detection antibody working solution to each well Incubate 10 minutes at RT ___ Following the detection antibody step dispense 5 uL OptiWash to each well Incubate 10 minutes at RT ___ Following the wash step dispense 5 uL of the SAv HRP working solution to each well Incubate 10 minutes at RT ___ Following the SAv HRP incubation dispense 30 uL OptiWash to each well Incubate 10 minutes at RT ___ Immediately following step 10 dispense 30 uL OptiWash to each well for a second 30 uL wash Incubate 10 minutes at RT ___ Dispense 10 uL OptiGlow working solution to each well Incubate 15 minutes at RT
9. tance is available under the Technical Support tab on the Siloam Biosciences web site http www siloambio com Material Safety Data Sheets MSDS Using Optimiser Immunoassay Microplate Video Optimiser User s Guide Reader Settings Quick Reference Guide Frequently Asked Questions Application Notes Two additional videos appear under the Technology tab of the web site Optimiser Principles of Operation Running an Assay with Optimiser DOC ID ETS 1 MS 0044 A1 QuantaRed substrate is supplied by Thermo Fisher Scientific Inc Page 12 of 13 SILOAM biosciences Better Immunoassays Through Innovative Microfluidics SILOAM BIOSCIENCES INC 413 Northland Blvd Cincinnati OH 45240 USA Tel 1 513 429 2976 Fax 1 513 429 2976 http www siloambio com
10. ts respectively b Note Prepare the working substrate solution no more than 30 minutes before reading the plate Page 4 of 13 Use of OptiBlock This method was developed using Siloam Biosciences OptiBlock not only as blocking agent following the coating of the microfluidic reaction chamber with capture antibody but also as diluent for the anti rabbit IgG HRP conjugate R amp D Systems Reagent Diluent was used as diluent for the standards and detection antibody The intent in developing this method was to demonstrate the ease of transitioning Invitrogen s AKT pS473 Antibody Pair from their intended use in conventional ELISAs to an Optimiser based ELISA format Use of this method for the analysis of tissue culture supernatant serum plasma or other sample types may require further optimization of some assay parameters by the investigator to achieve desired results for example standard curve and sample diluents Siloam Biosciences has developed OptiMax Standard Diluent for use in the analysis of cell culture supernatants This product has been incorporated in Siloam Biosciences commercially available OptiMax ELISA Kits Page 5 of 13 DISPENSING MATERIALS TO THE OPTIMISER PLATE 1 Optimiser assembly Assemble the Optimiser plate pad and holder as illustrated a Position the holder on the lab bench with the Optimiser logo facing the user b The absorbent pad must be positioned with the pl
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