Human Cytokine Antibody Array 1

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Antibody Array (AA), Multiplex ELISA
Pubmed 21 references available
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Quantity 1 kit
Quantity Comment 2 Samples
Shipping to United States ( )
Availability Will be delivered in 5 to 7 Business Days
Brand RayBio®
Specificity Detects: G-CSF, GM-CSF, GRO, GRO-alpha, IL-1 alpha, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-15, IFN-gamma, MCP-1, MCP-2, MCP-3, MIG, RANTES, TGF-beta1, TNF-alpha, TNF-beta
Characteristics RayBio® Human Cytokine Antibody Array 1 (2 membrane arrays) with Accessories, for simultaneous detection of 23 Cytokines in 2 samples. Suitable for all sample types
Components Human Cytokine Antibody Array membranes (2/4/8 membranes). Biotin-Conjugated Anti-Cytokines (1/2/4 tubes, each tube for two membranes). 1,000X HRP-Conjugated Streptavidin (1 tube). 1X Blocking Buffer (25/50 ml). 20X Wash Buffer I (10/20 ml). 20X Wash Buffer II (10/20 ml). 2X Cell Lysis Buffer (10/20 ml). Detection Buffer C (1.5/2.5 ml). Detection Buffer D (1.5/2.5 ml). Eight-Well Tray (1 each). Manual.
Material not included Small plastic boxes or containers. Orbital shaker. Plastic sheet protector or SaranWrap. Kodak X-Omat AR film (REF 165 1454) and film processor or Chemiluminescence imaging system
Background All cell functions, including cell proliferation, cell death and differentiation, as well as maintenance of health status and development of disease, are controlled by a multitude of genes and signaling pathways. New techniques such as cDNA microarrays have enabled us to analyze global gene expression 1-3 . However, almost all cell functions are executed by proteins, which cannot be studied simply through DNA and RNA techniques. Experimental analysis clearly shows a disparity between the relative expression levels of mRNA and their corresponding proteins 4 . Therefore, analysis of the protein profile is critical. Currently, two-dimensional polyacrylamide SDS page coupled with mass spectrometry is the mainstream approach to analyzing multiple protein expression levels 5,6 . However, the requirement of sophisticated devices and the lack of quantitative measurements greatly limit their broad application. Thus, effective study of multiple protein expression levels has been complicated, costly are time-consuming until now. Our Human Cytokine Antibody Arrays are the first commercially available protein array system 7-11 . By using the system, scientists can rapidly and accurately identify the expression profiles of multiple cytokines in several hours inexpensively. The kit provides a simple array format, and highly sensitive approach to simultaneously detect multiple cytokine expression levels from conditioned media, patient's sera, cell lysate, tissue lysates and other sources. Traditionally, cytokines are detected by using ELISA. However, This approach has several advantages over ELISA. First, and most importantly, our approach can detect many cytokines simultaneously. Secondly, sensitivity is greatly increased. As little as 4 pg/ml of MCP-1 can be detected using the protein array format. In contrast, at least 40 pg/ml of MCP-1 is required to produce a clear signal in an ELISA assay. Furthermore, the detection range is much greater than ELISA. For example, the detection range of IL-2 varies from 25 to 250,000 pg/ml using technology, whereas the detection range varies only within 100-1000 fold in a typical ELISA. Therefore, the detection range is greater with protein array compared with ELISA. The variation is lower than ELISA as well. As determined by densitometry, the variation between two spots ranged from 0 to 10% in duplicated experiments. In contrast, variation (about 20%) in ELISA is much higher. Finally, the system is much quicker and can be much easier to adapt to high-throughput technique. Pathway-specific array systems allow investigators to focus on the specific problem and are becoming an increasingly powerful tool in cDNA microarray systems. The first protein array system, known as Human Angiogenesis antibody array, is particularly useful in comparison with the human cytokine cDNA microarray system. Besides the ability to detect protein expression, the system is a more accurate reflection of active cytokine levels because it only detects secreted cytokines, and no amplification step is needed. Furthermore, it is much simpler, faster, environmentally friendlier, and more sensitive. Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool to study cytokines. Cytokines play an important role in innate immunity, apoptosis, angiogenesis, cell growth and differentiation 12 . Cytokines are involved in most disease processes, including cancer and cardiac diseases. The interaction between cytokines and the cellular immune system is a dynamic process. The interactions of positive and negative stimuli, and positive as well as negative regulatory loops are complex and often involve multiple cytokines. Without doubt, simultaneous detection of multiple cytokines provides a powerful tool to study cytokines.
Application Notes 1. Place each membrane into the provided eight-well tray (- mark is on the side printed with antibodies). 2. Add 2 ml 1X Blocking Buffer and incubate at room temperature for 30 min to block membranes. Make sure there are no bubbles between the membranes. 3. Decant Blocking Buffer from each container, and incubate membranes with 1 ml of sample at room temperature for 1 to 2 hours. Dilute sample using 1X Blocking Buffer if necessary. Note: Incubation may be done at 4°C for overnight. 4. Decant the samples from each container, and wash 3 times with 2 ml of 1X Wash Buffer I at room temperature with shaking. Please allow 5 min per wash. Dilute 20X Wash Buffer I with H 2 O. 5. Wash 2 times with 2 ml of 1X Wash Buffer II at room temperature with shaking. Allow 5 min per wash. Dilute 20X Wash Buffer II with H 2 O. 6. Prepare working solution for primary antibody. Add 100 µl of 1x blocking buffer to the Biotin-Conjugated Anti- Cytokines tube. Mix gently and transfer all mixture to a tube containing 2 ml of 1x blocking buffer. Note: the diluted biotin-conjugated antibodies can be stored at 4°C for 2-3 days. 7. Add 1 ml of diluted biotin-conjugated antibodies to each membrane. Incubate at room temperature for 1-2 hours. Note: incubation may be done at 4°C for overnight. 8. Wash as directed in steps 4 and 5. 9. Add 2 ml of 1,000 fold diluted HRP-conjugated streptavidin (e.g. add 2 µl of HRP-conjugated streptavidin to 1998 µl 1X Blocking Buffer) to each membrane. Note: mix the tube containing 1,000X HRP-Conjugated Streptavidin well before use since precipitation may form during storage. 10. Incubate at room temperature for 2 hours. Note: incubation may be done at 4°C for overnight. 11. Wash as directed in steps 4 and 5.
Sample Preparation Use serum-free conditioned media if possible. If serum-containing media is required, use an uncultured media aliquot as a negative control sample, since many types of sera contain cytokines. For cell lysates and tissue lysates, we recommend using Cell Lysis Buffer to extract proteins from cell or tissue (e.g. using homogenizer). Dilute 2X Cell Lysis Buffer with H 2 O (we recommend adding proteinase inhibitors to Cell Lysis Buffer before use). After extraction, spin the sample down and save the supernatant for your experiment. Determine protein concentration. We recommend using per membrane: 1 ml of Conditioned media (undiluted), or 1 ml of 2-fold to 5-fold diluted sera or plasma, or 50-500 µg of total protein for cell lysates and tissue lysates (use ~200-250 µg of total protein for first experiment) Dilute the lysate at least 10 fold with 1 X blocking buffer. Note: The amount of sample used depends on the abundance of cytokines. More of the sample can be used if the signals are too weak. If the signals are too strong, the sample can be diluted further. o If you experience high background, you may further dilute your sample. B. Handling Array Membranes. Always use forceps to handle membranes, and grip the membranes by the edges only. Never allow the array membranes to dry during experiments. C. Incubation. Completely cover the membranes with sample or buffer during incubation, and cover the eight-well tray with lid to avoid drying. Avoid foaming during incubation steps. Perform all incubation and wash steps under gentle rotation. Several incubation steps such as step 2 (blocking), step 3 (sample incubation), step 8 (biotin-Ab incubation) and step 11 (HRP-streptavidin incubation) may be done at 4°C for overnight, but make sure to cover the 8 well plate tightly to prevent evaporation
Assay Procedure Do not let the membrane dry out during detection. The detection process must be completed within 40 minutes without stopping. 1. Proceed with the detection reaction. Add 250 µl of 1X Detection Buffer C and 250 µl of 1X Detection Buffer D for one membrane, mix both solutions, Drain off excess wash buffer by holding the membrane vertically with forceps. Place membrane protein side up (- mark is on the protein side top left corner) on a clean plastic sheet (provided in the kit). Pipette the mixed Detection Buffer on to the membrane and incubated at room temperature for 2 minute. Ensure that the detection mixture is completely and evenly covers the membrane without any air bubbles. 2. Drain off any excess detection reagent by holding the membrane vertically with forceps and touching the edge against a tissue. Gently place the membrane, protein side up, on a piece of plastic sheet (- mark is on the protein side top left corner). Cover another piece of plastic sheet on the array. Gently smooth out any air bubble. Avoid using pressure on the membrane. 3. Expose the array to x-ray film (we recommend to use Kodak X-Omat AR film) and detect the signal using film developer, or the signal can be detected directly from the membrane using a chemiluminescence imaging system. . Expose the membranes for 40 Seconds. Then re-expose the film according to the intensity of signals. If the signals are too strong (background too high), reduce exposure time (e.g. 5-30 seconds). If the signals are too weak, increase exposure time (e.g. 5-20 min or overnight. Or re-incubate membranes overnight with 1X HRP-conjugated streptavidin, and redo detection in the second day. 4. Save membranes in -20°C to -80°C for future references.
Restrictions For Research Use only
Storage -20 °C
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