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6737 Mowry Ave
Newark, CA 94560
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Registration enables users to use special features of this website, such as past
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LSBio Phospho-Specific ELISA Kits
LifeSpan offers a number of different types of ELISA kit that are specific for the detection of phosphorylated target proteins.
Phospho-specific kits are available for standard ELISA detection, in vitro detection in adherent cells, and detecting transcription
factor activity in nuclear extracts and cell lysates. Each kit is ready-to-use with all the necessary reagents and a clear, concise
protocol that will step you through the process, from sample preparation to analysis of the results.
Phospho-Specific Sandwich ELISA Kit Protocol
| 1) The supplied 96-well microtiter plate is pre-coated either pan- or phospho-specific capture antibody. |
| 2) Control standards or test samples are added to the well and the target antigen is captured by the antibodies. |
| 3) Phospho-specific or pan-specific primary antibody is added and binds to the target antigen. |
| 4) The wells are washed and a biotinylated secondary antibody is added and binds to the primary antibody. |
| 5) The wells are washed and a solution containing HRP-conjugated Avidin is added. The Avidin binds to the biotinylated secondary antibody. |
| 6) The wells are washed and a solution containing TMB is added. The TMB reacts with the HRP changing from colorless to a blue color. An acid stop solution is then added, the reaction stops, and the blue color changes to yellow. |
Phospho-Specific DNA-Binding ELISA Kit Protocol
| 1) The 96-well microtiter plate comes pre-bound with specific double-stranded (dsDNA) oligonucleotides. |
| 2) Following a blocking step samples are added to each well and phosphorylated transcription factor binds to the oligonucleotides. |
| 3) The wells are washed and a phospho-specific primary antibody is added which binds activated transcription factor. |
| 4) The wells are washed and a HRP-conjugated secondary antibody is added which binds to the primary antibody. |
| 5) The wells are washed and a solution containing TMB is added. The TMB reacts with the HRP changing from colorless to a blue color. An acid stop solution is then added, the reaction stops, and the blue color changes to yellow. |
Phospho-Specific Cell-Based ELISA Kit Protocol
| 1) Cell monolayers are cultured in the 96-well microtiter plate. Each well can then be treated with stimulants, inhibitors, etc. to induce expression of the phosphorylated target. The cells are then fixed and blocked. |
| 2) Phospho-specific or pan-specific primary antibody is added and binds to the target antigen on the cells surface. |
| 3) The wells are washed and a HRP-conjugated secondary antibody is added which binds to the primary antibody. |
| 4) The wells are washed and a solution containing TMB is added. The TMB reacts with the HRP changing from colorless to a blue color. An acid stop solution is then added, the reaction stops, and the blue color changes to yellow. |
The plate can then be read in a spectrophotometer at a wavelength of 450 nm.
