Arrayit Human Total IgG Microarray Controls (Set of 3) contain 1.0 ml of human total immunoglobulin G (IgG) whole molecule antibody at concentrations of 0.1, 0.01 and 0.001 µg/µl final concentration in microarray printing buffer for research assays that measure human IgG antibody levels. This product can be used as a positive control or as an internal assay control with an anti-human IgG secondary antibody or staining reagent in either a fluorescent or colorimetric format. Arrayit Human Total IgG Microarray Controls (Set of 3) arrive pre-mixed and ready to use with an Arrayit microarray printer or another suitable microarrayer. This product ships frozen on cold packs. Please refrigerate or freeze up receipt.
Amplification and Labeling - Arrayit Human Total IgG Microarray Controls (Set of 3) to Quantify Human IgG Antibody Levels in Research, Life Sciences and Blood Testing Applications
Arrayit Human Total IgG Microarray Controls (Set of 3) provide a set of controls that can be used in microarray testing applications requiring positive controls for human IgG levels. The IgG controls contain 1.0 ml of human total immunoglobulin G (IgG) whole molecule antibody at concentrations of 0.1, 0.01 and 0.001 µg/µl final concentration in microarray printing buffer for research assays that measure human IgG antibody levels. This product can be used as a positive control or as an internal assay control with an anti-human IgG secondary antibody or staining reagent in either a fluorescent or colorimetric format. Arrayit Human Total IgG Microarray Controls (Cat. IGG) arrive on cold packs, pre-mixed and ready to use with an Arrayit microarray printer or another suitable microarrayer. Please refrigerate at 4°C for regular use or freeze at -20°C for extended storage.
.
Figure 1.Arrayit Human Total IgG Microarray Controls (Set of 3) are packaged in an anti-static shipping envelop as shown. Cut just below the heat seal at the top of the envelop to open and re-seal after use. The human total IgG controls are 0.1, 0.01 and 0.001 µg/µl final concentration to provide positive controls over 100-fold range at 10-fold increments. These controls will provide strong positive signals in assays that use an anti-IgG fluorescent or staining secondary reagent to measure total IgG levels.
Table of Contents
- Introduction
- Quality Control
- Product Description
- Short Protocols
- Complete Protocols
- Troubleshooting Tips
- Technical Support
- Scientific Publications
- Ordering Information
- Warranty
Introduction
Congratulations on taking a big step towards improving the affordability, quality and speed of your genomics, biomedical, pharmaceutical and agricultural research. This booklet contains all the information required to take full advantage of Arrayit Human Total IgG Microarray Controls (Set of 3).
Quality Control
Arrayit takes every measure to assure the quality of our Arrayit Human Total IgG Microarray Controls (Set of 3). The finest microarray biochip research and engineering was used to develop this product line. Rigorous quality control monitoring on a lot-by-lot basis guarantees that this product conforms to the highest industry standards.
Product Description
Arrayit Human Total IgG Microarray Controls (Set of 3) provide the highest quality human total IgG positive controls at an affordable price. Our total IgG controls were developed by our team of world-class experts and are manufactured in our advanced microarray research laboratories by our trained technical staff.
Users will appreciate the following features:
1. Set of three human total IgG positive controls
2. 1.0 ml volume of whole molecule human IgG per control
3. 0.1, 0.01 and 0.001 µg/µl final concentration
4. Can be used in all assays that measure human total IgG levels
5. Arrive pre-mixed and ready to use
6. Stable for one year from the date of receipt
7. Protein microarray, life sciences and microarray testing applications
8. Provide strong positive signals with human anti-IgG fluorescent secondary reagents
9. Provide strong positive signals with human anti-IgG colorimetric secondary reagents
10. Compatible with contact and non-contact microarray printers
Short Protocol
1. Open the silver anti-static envelop.
2. Remove the three controls (1.0 ml each) and mix gently by inverting.
3. Centrifuge briefly (Cat. MHC110V) to consolidate the liquid samples.
4. Pipette 10 µl into 384-well microplate (Cat. MMP384).
5. Print into microarrays using a microarray printer.
6. React with a blood specimen containing human IgG.
7. Stain the microarray to detect IgG binding.
8. Scan the microarray to capture the fluorescent or colorimetric signals.
9. Quantify the data.
10. Verify positive IgG signals with the Arrayit controls.
Complete Protocol
1. Open the silver anti-static envelop. Use a scissors to cut the silver anti-static envelop just below the heat seal at the top of the envelop. Make sure to cut above the zip-lock for resealing. The controls are ready to use immediately. If the controls are to be used on a monthly basis or more often, store in a refrigerator at 4°C. If the controls are to be used less than once per month, store in a freezer at -20°C. Arrayit Human Total IgG Microarray Controls (Set of 3) are good for one year from the date of receipt.
2. Remove the three controls (1.0 ml each) and mix gently by inverting. If the controls are frozen from -20°C storage, gently thaw the controls for 10 min at room temperature. Prior to use, the tubes should be inverted several times by hand to ensure proper mixing.
3. Centrifuge briefly (Cat. MHC110V) to consolidate the liquid samples. Each of the three controls contains 1.0 ml of liquid volume comprising human total IgG in a physiologic phosphate buffer. Please the three tubes in an Arrayit High-Speed Centrifuge (Cat. MHC110V) or equivalent and centrifuge for a few seconds to consolidate the liquid in the bottom of the tube. This ensures that the controls will not spill when opening the screw cap lid.
4. Pipette 10 µl into 384-well microplate (Cat. MMP384). Arrayit Human Total IgG Microarray Controls (Set of 3) are used as positive controls by plating these specimens in the same microplate that contains the other protein specimens used in the test. Pipette 10 µl of each of the three controls in a separate well of a 384-well microplate. The controls are ready to print and should be used undiluted at the specified 0.1, 0.01 and 0.001 µg/µl final concentrations. Make sure to program proper pin washing steps into the manufacturing routine to prevent contamination of the other protein specimens with trace levels of the IgG positive controls.
5. Print into microarrays using a microarray printer. Manufacture the microarrays containing all of the specimens in the microplate including the three Arrayit total IgG positive controls. Multiple technical replicates such as duplicate or triplicate printed spots of each specimen can be used to increase quantification in quantitative assays. After printing and just before use, make sure to activate the microarrays using Arrayit Microarray Activation Buffer (Cat. PMAB), which removes unbound printed samples and blocks the microarray surface to produce high signal strength and low background.
6. React with a blood specimen containing human IgG. After printing, the microarrays can be used to detect human IgG molecules in whole blood and other types of biological specimens. React all of the microarray spots including the test and Arrayit IgG positive control specimens to obtain proper results. For best results, use Arrayit Protein Microarray Reaction Buffer (Cat. PMRB). Gentle mixing of the hybridization cassette or reaction chamber generally produces superior results compared to static reactions. A 30-60 minute reaction time is generally sufficient to allow binding of human IgG to the reactive proteins comprising the printed microarrays.
7. Stain the microarray to detect IgG binding. After the reactive step, wash the microarrays thoroughly to remove unbound IgG molecules. Proper washing will ensure low and uniform background in the assay. After the wash steps, stain the microarrays with a reagent that will bind to the IgG molecules present on the microarray. Suitable staining reagents include fluorescent secondary reagents such as human anti-IgG reagents that contain fluorescent tags and colorimetric reagents with bound enzymes. Both types of reagents will detect printed test specimens that contain bound human IgG as well as the three Arrayit IgG positive controls. A 30-60 minute staining time is generally sufficient to allow binding of human anti-IgG staining reagent to IgG molecules on the reacted microarrays.
8. Scan the microarray to capture the fluorescent or colorimetric signals. After the staining step, make sure to wash the microarrays thoroughly to remove all unbound staining reagent to ensure low and uniform background. After the wash steps, rinse the microarray slide briefly using Protein Microarray Rinse Buffer (Cat. PMNB) and dry the slide using an Arrayit Microarray High-Speed Centrifuge (Cat. MHC110V). Insert the slide in an Arrayit InnoScan Microarray Scanner and scan at the laser and photomultiplier tube settings that are appropriate for the assay. For most applications, scanner settings that produce saturated signals at 1% of the microarray spots provide a good starting point. Saved the scanned microarray image as a 16-bit TIFF file.
9. Quantify the data. Use Arrayit MAPIX Microarray Quantification and Analysis software (Cat. MAPIX) to open the microarray TIFF file containing the scanned fluorescent or colorimetric data. Place a software grid around each spot on the microarray and quantify the data. Save the quantified microarray data as a .txt file for further analysis. The .txt data can be rendered into a test report showing results for each printed test specimen and the Arrayit IgG positive controls.
10. Verify positive IgG signals with the Arrayit controls. In the quantified data, make sure that the three Arrayit human IgG positive controls (Cat. IGG) produce positive signals. The 0.1, 0.01 and 0.001 µg/µl positive controls should produce decreasing intensity signals corresponding to the 10-fold dilutions of the three controls. Positive signals for the Arrayit IgG positive controls indicate that all of the main processing steps (printing, activation, reaction, washing, rinsing, drying, scanning and quantification) worked properly. The IgG control signals can also be used to add quantification for the test specimens as follows. The 0.1 µg/µl IgG control printed with Arrayit SMP3B pins will deposit a printed droplet of 0.9 nanoliters (0.9 nl), which will contain 9 x 10^-5 µg of human IgG protein per printed microarray spot. Assuming 30% coupling efficiency to the microarray slide after printing, the 0.1 µg/µl IgG positive control spot will contain 2.7 x 10^-5 µg of human IgG protein. IgG protein quantity can be converted to the number of IgG molecules using the molecular weight of IgG (150,000 grams/mole) and Avogadro's Number (6.02 x 10^23 molecules/mole). Each microarray spot printed with the 0.1 µg/µl IgG positive control will contain 1.8 x 10^-16 moles of IgG, which equals 1.1 x 10^8 IgG molecules. A printed microarray test spots that produces the same signal as the 0.1 µg/µl control would therefore contain 1.1 x 10^8 IgG molecules bound to the reactive antigens in the test spot. This calculation can be repeated for the 0.01 and 0.001 controls to create a standard curve based on concentration and signal intensity. Test proteins on the microarray can then be plotted on the standard curve to provide a semi-quantitative measure of IgG bound to each microarray test spot.
Troubleshooting Tips
1. No signals from positive controls
- Verify the activity of the staining reagent
- Make sure that the microarray samples were printed properly
2. Saturated positive control signals
- Reduce the concentration of the staining reagent
- Reduce the scanner settings
3. Non-linear signals observed across the controls
- Adjust the scanner settings up or down
- Make sure to dry to microarrays after printing to ensure efficient coupling of the IgG positive controls to the surface