Immunome TM Protein Array

Transcription

1 Immunome TM Protein Array User Manual Last Revised, September 7 th 2016 Product code: 9PAH-IMM-1631 ISO & GLP Certified Tel: (Toll Free) or ; Fax: Web: info@raybiotech.com

2 RayBiotech, Inc. Immunome TM Protein Array Manual Protocol TABLE OF CONTENTS Page I. Introduction 2 II. Immunome Technology 2 III. How it Works 5 IV. Materials provided 6 V. Additional Materials and Equipment Required 6 VI. Experimental Considerations 7 VII. Reagent Preparation 7 VIII. Protocol 8 a. Preparation of assay solutions 8 b. Assay set up 9 c. Washing after serum binding 10 d. Incubation with Cy3-antiIgG 10 e. Washing after incubation with Cy3-anti IgG 11 IX. Array Scanning and Image Analysis 12 2

3 I. INTRODUCTION The unique aspect of this technology is that only full-length, correctly folded, functional proteins are able to be arrayed on the slide surface. The technology relies on the use of a proprietary (BCCP) affinity tag which monitors folding. Autoantibodies, which are raised when the immune system responds to the body's own proteins, are produced in response to many diseases, including cancer, autoimmune disorders and neurodegenerative conditions. These autoantibodies often precede disease symptoms making them highly effective tools for disease diagnosis and stratification. The Immunome Protein Array allows detection of clinically relevant autoantibodies in patient serum samples. II. IMMUNOME TECHNOLOGY Proteins are by nature diverse in size, structure and function. Tagging proteins with BCCP offers several advantages in array fabrication. The protein is biotinylated during natural processing, but only if it is correctly folded. Also BCCP presents the protein at a distance of some 50Å from the slide surface so assay reagents have good access to active sites on the immobilized proteins. Such benefits are essential to ensuring that data from Immunome are reliable and meaningful. Immobilization of proteins on to any surface is fraught with dangers, particularly if the mode of attachment of the protein to the surface is not tightly controlled. By contrast, immobilization of proteins via an affinity tag is a technique employed routinely during the course of affinity purification of proteins prior to functional analysis so there is ample precedent to suggest that, providing the affinity tag itself is tolerated, the process of immobilization on to a surface via the tag does not generally affect protein folding or function. Immunome therefore attaches each arrayed protein to the surface exclusively via an affinity tag, specifically the biotin carboxyl carrier protein (BCCP) domain of the E. coli acetyl CoA carboxylase. Biotinylation in vivo of BCCP does have one constraint; the BCCP domain must be folded into its native 3-dimensional structure in order to become biotinylated. Indeed short peptides derived from BCCP which contain that linear biotinylation epitope are not recognized as substrates by the biotin ligases. This property allows the biotinylation state of BCCP to be used as a folding marker. Most studies to date have focused on fusion proteins to BCCP where the fusion partner is an intact domain that is known to fold efficiently in the host cell. In such cases, it is easy to imagine that each domain in the fusion protein will fold autonomously, and that the resultant fusion protein will appear like beads on a string, allowing the BCCP domain to become biotinylated post-folding. However, when eukaryotic proteins are expressed in Immunome TM Protein Array User Manual 3

4 heterologous hosts, it is not automatic that they will fold correctly. If they do not, there is a strong likelihood that the aberrant folding of the fusion partner will affect the downstream biotinylation of BCCP, particularly if the BCCP domain lies at the C-terminal end of the fusion protein, as it does in the Immunome s recombinant proteins. If the expressed protein mis-folds, the correct folding, and therefore biotinylation, of the BCCP tag is prevented. By contrast if the protein folds correctly then folding of the BCCP tag should be unhindered and biotinylation will result. Consequently it is likely that only correctly folded proteins will be immobilized on to the array surface. The logic behind this result is as follows. During translation of large proteins, folding almost certainly begins in a co-translational manner this is known for eukaryotic systems and is likely to be true in prokaryotic systems, although there are some differences here due to the chaperone-like roles played by heat shock proteins during initial protein synthesis. Cotranslational folding of a fusion partner means that in the majority of cases, the N-terminal domain will start to fold first. If this initial folding event results in a properly folded domain, then it is reasonable to suppose that the C-terminal domain will also be able to fold correctly (unhindered by the presence of the N-terminal fusion). However, if the N-terminal domain mis-folds and starts to present aberrant hydrophobic surfaces, there is a strong likelihood that initiation of the normal folding pathway of the C-terminal domain will be affected, leading to aberrant folding of the C-terminal domain as well. If the C-terminal domain is the BCCP domain, it would be expected that mis-folding of an N-terminal fusion partner will perturb the proper folding of BCCP and therefore prevent downstream biotinylation of BCCP. 4

5 III. HOW IT WORKS Autoantibody detection A panel of correctly folded, functional proteins is printed in 4 identical arrays on a standard glass microarray slide. After a blocking step, samples are incubated with the arrays. Nonspecific auto-antibodies and other molecules are then washed off, and the arrays are incubated with Cy3 labelled anti-human IgG detection antibodies. Signals are then visualized using a fluorescence laser scanner. The 1631 Immunome Array requires 1 full slide for 1 sample. To view How It Works for other applications (Small Molecule-Kinase Inhibition, Protein- Protein Interaction, DNA Binding, and Methylation), click the link below: Immunome TM Protein Array User Manual 5

6 IV. MATERIALS PROVIDED Item Code Material/Reagent Quantity Included for 9PAH-IMM-1631-X 1 Slide (-1) 2 Slide (-2) 4 Slide (-4) 8 Slide (-8) Purpose Immunome slides Quadruplicate printed Immunome Protein Array Cy 3 Labeled IgG 35 μl 35 μl 65 μl 125 μl Secondary antibody Pooled Normal Serum 30 μl 30 μl 30 μl 30 μl Positive control Quadriperm Dish Incubation Pap Jars Washing / Incubation User manual 1 Instructions Gal file (upon request) 1 Data analysis V. ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED Serum Assay Binding (SAB) Buffer (see Page 6) High purity MilliQ water 15 ml Centrifuge tubes Kimwipe tissues (or similar) Nitrile gloves Tube racks to hold 50 ml centrifuge tubes Incubated Shaker Shaker Vortex Lab pipette (10-50 ml) Lab pipette (1-5 ml) 200 µl & 100 µl pipette Centrifuge with 13,000 rpm Volumetric flask Microarray Scanner (Agilent-G2505C / Inopsys-710 AL / Genepix 4000B, or similar) See a list of potential scanners here on our website: 6

7 VI. EXPERIMENTAL CONSIDERATIONS Please make sure you wear gloves before handling any reagents or arrays. Handle the slides by holding the area with barcode labels, or side edges only. Do not touch the slide surface. Please note that any variation in buffers, pipetting technique, washing technique, incubation time or temperature can alter the performance of the kit. Only use reagents and materials recommended by this user s guide. Do not substitute buffers or solutions from other sources, and make fresh buffers when possible. Do not allow arrays to dry out between incubation and washing, as this can cause high background. Slides should be scanned as soon as possible after the array is completed. If scanning cannot be done immediately, store the slide in a dark, low humidity environment at 4 C for up to 4 weeks at most. If sending to RayBiotech for our free scanning service, please contact RayBiotech Technical Support ASAP to discuss your needs and get the slides prepared. TechSupport@RayBiotech.com VII. REAGENT PREPARATION SAB Buffer is not provided in the kit, and should be made prior to the running of the assay. It is advisable to use freshly prepared buffer to run the assay. However, SAB Buffer can be stored at 4 C for up to one week. A total of 250mL Serum Assay Binding (SAB) Buffer is required to run each sample to ensure you have enough volume for all the needed steps. For more samples, plan to make additional buffer. For Example: 5 samples require 1.25L of total SAB Buffer (250mL * 5 samples). SAB Buffer (1 liter): Dissolve 1 g of Bovine Serum Albumin (BSA), 1 ml of Triton-X and 100 ml of 10 x Phosphate Buffered Saline (PBS) in 899 ml of deionized water. Store at 4 C until use. Immunome TM Protein Array User Manual 7

8 VIII. PROTOCOL Below is the protocol for screening protein-antibody interactions. Protocol can be adapted for small molecule-kinase inhibition, protein-protein interactions, or DNA-protein binding assays (contact technical support for more information). A. Preparation of Assay Solutions (serum sample dilution) 1. Ensure that all materials are ready and labelled prior to removing the serum samples from the freezer. 2. Once ready to start the assay, remove the serum samples from the freezer. Note: Do not allow the samples to thaw during this process in case they need to be replaced in the freezer. 3. Check each sample visually to ensure that each of the tubes has sufficient serum (22.5 μl) for assay. Number the tube(s) to be run, and place them on ice to thaw for 30 min. 4. When completely thawed, vortex to mix each sample at full speed for 3-5 seconds. 5. Place the tubes in a microcentrifuge. Centrifuge for 3 minutes at 13,000 rcf. a. Disinfect the centrifuge with 70% ethanol if spillages occur. 6. Starting with sample number 1, remove the tubes from the centrifuge and place in a sample tube rack. 7. The serum samples must now be diluted in SAB buffer to provide the assay solutions. Since these are potentially infectious samples, we recommend this be performed in a class II Biological safety cabinet. Be careful not to disturb the undiluted serum samples. 8. Number 15mL falcon tubes, one per sample, and add 4.5mL of SAB buffer to each. 9. Starting from number 1 in the assay rack, remove the first sample and pipette 22.5 μl of the sample into its numbered 15mL tube with 4.5 ml of SAB Buffer. 10. Vortex to mix each sample at full speed for 3-5 seconds. Place the vortexed tube in the same position in a second tube rack to avoid confusion with unused buffer tubes. 11. Follow the same procedure for the remaining samples (if needed), transferring each sample tube and buffer tube after each pipetting operation. 12. If you want to use the included positive control (pooled normal serum), you can dilute this in the same manner as the samples. This will serve as a reference/standard if run, but will require an additional slide to perform, so plan accordingly. 8

9 B. Assay Set Up 13. Take the Pap Jars (Item 30018) from the box, and add 30 ml cold SAB from the fridge. You will need one Pap Jar with 30mL cold SAB for every 2 slides to be run. SAB Buffer can now be stored at room temperature for the remainder of the assay. 14. Take each Immunome Slide (Item 30014) out from the tube containing their storage buffer using forceps. Grip the array between finger and thumb at the labelled end of the slide, or along the edges of the slide, to avoid touching the array. 15. Drain excess liquid from the array by touching the edge of the array on the rim of the Pap Jar, or lightly onto a Kimwipe. 16. Carefully add the slide to the Pap Jar containing 30mL cold SAB. 17. Up to 2 slides can be added to each Pap Jar with 30mL of cold SAB. 18. When all the arrays have been added to the Pap Jars, ensure the lid is closed tightly and gently shake the Pap Jars up and down five times to aid mixing at the slide buffer interface. 19. Place the Pap Jar on an orbital shaker to shake 50 rpm, for 5 minutes. 20. Near the end of the slide wash step (Step 19), pipette 4.0 ml of each diluted sample (Step 9) into a numbered chamber in the Quadriperm dishes (Item 30017) to create a Sample Probing Solution. 21. Place several layers of white paper toweling onto the bench surface and cover this with three layers of Kimwipe tissues. 22. When the slides have finished washing, carefully pour off the cold SAB and remove each array from the SAB Buffer one at a time. 23. Grip the slide at the labelled end between finger and thumb and wipe the back of the array once on the Kimwipe tissue paper, then blot the long edge of the array three times on the wad of lint free tissue paper. 24. Place the array immediately into the Sample Probing Solution in the correct numbered chamber of the Quadriperm dish. Ensure that the array does not rest on the square plastic lugs at the bottom of the plate. 25. After addition of the first array slide, set a timer to countdown for 2 hours. Gently swirl the plate to cover the slide with incubation solution. 26. After addition of all slides, record the barcode number to track them, or scan the barcode on each slide if applicable. 27. Replace the lids of the Quadriperm dishes and incubate in the shaking incubator at 50 rpm, 20 C for 120 min. Tip: Ensure that the dishes are kept horizontal at all times to prevent mixing of solutions between chambers. Handle the dishes very gently to prevent mixing or splashing of contents between chambers. Immunome TM Protein Array User Manual 9

10 C. Washing after Serum Binding 28. Towards the end of the incubation period, fill and number the needed amount of Pap Jars (Item 30018) with 30 ml of SAB (1 Pap Jar needed per sample). 29. When the incubation time has finished, remove each array from the Quadriperm dishes and place into individually numbered Pap Jars. 30. Wash 1: Tip the dish slightly to move the arrays away from the numbered end of the dish and use a spatula or forceps to lift the array at the numbered side of the dish. Grip the labelled end of the slide between finger and thumb and place the first slide into its numbered and buffer filled Pap Jar. Tip: make sure that your gloves are clean before handling arrays directly. 31. Close the Pap Jar tightly and invert 4 times before placing on an orbital shaker. 32. Shake the Pap Jars at 50 rpm at 20 C. 33. Start a timer to countdown 20 min after addition of the first array to the buffer. 34. Process the remaining slides and place each in the orbital shaker at 50 rpm. 35. Wash 2: After the 20 min incubation has finished, take the first array and pour off the wash solution into an empty beaker then dispense another 30 ml of SAB into the tube at the back of the array. 36. Close the lid and invert the Pap Jar four times and place on the shaker at 50 rpm at room temperature. 37. Repeat for all remaining jars. Start the timer to countdown 20 min from the first jar. 38. Wash 3: After the 20 min incubation has finished, take the first array and pour off the wash solution into an empty beaker then dispense another 30 ml of SAB into the tube at the back of the array. 39. Close the lid and invert the Pap Jar four times and place on the shaker at 50 rpm at room temperature. 40. Repeat for all remaining jars. Start the timer to countdown 20 min from the first jar. D. Incubation with Cy3-anti IgG 41. Near the end of the last wash step (38-40), make up the Cy3-Anti-Human IgG probing solution. 42. To make up 30mL of Cy3-Anti-Human IgG probing solution, add 30mL of SAB solution at 20 C to a numbered Pap Jar, then add 30 µl of Cy3-anti-human IgG (Item 30015). Close the lid and mix well by repeated inversion. Add 30mL of this Cy3-anti-human IgG Probing Solution to a fresh Pap Jar. 10

11 43. 1 Pap Jar with 30mL of Cy3 Probing solution is enough to probe up to 2 slides. Make additional Pap Jars as needed with the above method (Step 42). 44. Place a wad of Kimwipe tissues on top of the working bench. Ensure that these do not become contaminated with buffer. 45. After the third wash is finished (Step 38-40), remove each slide carefully with forceps. Grip the slide at the labelled end between finger and thumb and wipe the back of the array once on the Kimwipe tissue paper, then blot the long edge of the array three times on the wad of lint free tissue paper. 46. If obvious residual buffer is still present, carefully touch the corner of the slide to the Kimwipes as well to remove this excess buffer. 47. Immediately place the arrays in the Cy3-anti-human IgG Probing Solution. 48. Close the list and invert the tube up and down five times to help mixing of the solution at the surface of the arrays. 49. Place the Pap Jars on a shaking incubator at 50 rpm, at 20 C incubate for 120 min. E. Washing After Incubation with Cy3-anti IgG 50. Towards the end of the incubation period, number a new Pap Jar and fill with 30mL SAB buffer at 20 C. 51. Wash 1: When the incubation has finished, grip the slide at the labelled end between finger and thumb and wipe the back of the array once on the Kimwipe tissue paper. Then blot the long edge of the array three times on the wad of lint free tissue paper, and place them into the Pap Jar with 30mL SAB wash solution. 52. Close the list tightly and invert slowly up and down five times, then place on a shaker for 5 min at 50 rpm at room temperature. 53. Wash 2: After wash 1 has finished, carefully pour off the SAB buffer, and replace with 30mL of fresh SAB buffer (20 C). 54. Close the list tightly and invert slowly up and down five times, then place on a shaker for 5 min at 50 rpm at room temperature. 55. Wash 3: After wash 1 has finished, carefully pour off the SAB buffer, and replace with 30mL of fresh SAB buffer (20 C). 56. Close the list tightly and invert slowly up and down five times, then place on a shaker for 5 min at 50 rpm at room temperature. 57. Wash 4: When the third wash has finished, carefully pour off the SAB buffer, and fill the Pap Jar with 200mL or MilliQ water. Immunome TM Protein Array User Manual 11

12 58. Close the list tightly and invert slowly up and down five times, then pour off the MilliQ water. 59. Wash 5 through 7: Repeat this MilliQ water wash step three times to ensure the buffer components are washed away from the Pap Jar and Slides. 60. Place a wad of Kimwipe tissue on the cleaned benchtop. 61. Remove the slides from the Pap Jars after the last pour off. Grip the slide at the labelled end between finger and thumb and wipe the back of the array once on the Kimwipe tissue paper, then blot the long edge of the array three times on the wad of Kimwipes. 62. Invert the washed Pap Jar onto the Kimwipes as well to remove as much residual liquid as possible before proceeding. 63. If obvious residual buffer is still present on the slides, carefully touch the corner of the slide to the Kimwipes as well to remove this excess buffer. 64. Place the slides back into the MilliQ washed Pap Jar (Step 62) and load into a centrifuge. 65. Dry the arrays by centrifugation for 2 min at 240 x g at 20 C. Note: Add a balancing tube if necessary 66. After centrifugation, place the lid in a biosafety hood with the lights off. Open the lid and allow the slide to air dry for 5-10 minutes. 67. Meanwhile, prepare the Microarray scanner, or any software (GAL file) or other materials needed to setup the machine. 68. Ideally the slide should be scanned immediately after the slide dries in Step 66. However, if sending the slide to RayBiotech, or scanning at a later date, the slide should be stored at 4 C, dark, and in a low humidity environment for no more than 2 weeks before being read. 12

13 IX. ARRAY SCANNING & IMAGE ANALYSIS All scanners that are compatible with 25 mm x 76 mm microscope slides can be used to scan Immunome protein arrays. Recommended Scanning Resolution: 10 μm Recommended scanners can be found on our website here: Special Note: RayBiotech can scan your slide for you if a scanner is not available. Please note this must be done ASAP, so prepare accordingly by contacting TechSupport@Raybiotech.com with any questions and needed paperwork. Image Analysis Array images can be analyzed by any image quantification software including analysis software provided by most scanner manufacturers, third-party software, or open source programs. A GAL (GenePix Array List) file for each array is generated to aid image acquisition. Please note GAL files are grid files specific to Genepix software and may not be compatible with any other software. GAL automatically generates grids on the array slide for auto spot detection supporting image analysis. Quality Control Different methods of quality control of both raw and normalized data must be done to verify the quality of the protein array data before proceeding with the data analysis: o Visualization of data using boxplot and normal distribution plot o Analysis of IgG serial dilution o Analysis of Cy3-BSA Data Analysis A wide range of different methods have been proposed for the analysis of microarray data. Here we present one example for identifying putative biomarkers based on expression ratios between case and control groups. 1. Calculate median of the raw signal intensities from the quadrupled gene spots on each slide (i.e. each sample): Immunome TM Protein Array User Manual X = (m 1 + m 2 + m 3 + m 4 ) m = signal intensity of replicates for each protein 13

14 X = Raw median for each gene in each sample 2. Subtract median background signals from the median raw signal intensities. 3. Normalize the data: K = X / <X> K = Normalized value <X> = median of all proteins in sample 4. Calculate fold change, FC Fold Change, FC = µcase / µcontrol 5. Volcano Plot Analysis (* for illustration purposes only) 14

15 This product is for research use only RayBiotech, Inc. Immunome TM Protein Array User Manual 15