|
Human Phospho-MAPK Array Kit FAQ
 |
Human Phospho-MAPK Array Kit FAQ |
Can other lysis buffers be used?
The kit has been validated for use with Lysis Buffer 6. We cannot guarantee
that the assay performance will be acceptable if other lysis buffers are used.
Can a non-film based image acquisition system, such as a cooled CCD camera, be used?
Yes. You will have to follow the instrument manufacturer's protocol
and optimize the exposure length.
Can the array be stripped and reused?
No. The arrays are validated for single-use only.
What exposure times should be used for chemiluminescent detection with X-ray film?
Levels of phosphorylation will vary with cell type and treatment. Multiple
exposures should be obtained in order to acquire as much information as possible
from each array experiment. We recommend exposures ranging from 1 minute to
10 minutes.
Can the sensitivity be increased by altering the Detection Antibody Cocktail or Streptavidin-HRP concentration?
Use the recommended concentrations of Detection Antibody Cocktail and Streptavidin-HRP.
Using a higher concentration of either may result in high background. Longer
exposures to X-ray film (5 to 10 minutes) should help to increase sensitivity.
What is the sensitivity of the Phospho-MAPK Array?
The Human Phospho-MAPK Array has a sensitivity better than, or comparable to, a Western blot.
Can tissue lysates be used?
Currently the kit is only validated for use with lysates prepared from cultured
cells. We are in the process of evaluating the use of tissue lysates with this
kit.
How is it known that the spotted capture antibodies are actually working? What samples were used for testing?
All antibodies were carefully selected using lysates from treated cell cultures
or recombinant proteins. Quality control testing is done on each lot using
a panel of lysates prepared from treated cell cultures.
I would like to confirm a positive signal for a kinase by IP-Western
blot using the capture antibody on the array. Can I inquire if the antibody
is listed in R&D Systems' catalog?
The identities of capture antibodies on the Phospho-MAPK Array are proprietary
information. However, we do sell antibodies for most of the kinases on the
array that are validated for Western blot applications. Strepatvidin-HRP is
also available (Catalog # DY998).
I would like to quantitate the phosphorylated kinases in the cell
lysates. Can this be done using normalized signals from the Human Phospho-MAPK
Array?
The Human Phospho-MAPK Array is best used as a screening tool to determine
the relative levels of kinase phosphorylation between untreated and treated
cell lysates. R&D Systems DuoSet IC ELISA Development Systems and Surveyor
IC ELISA Kits are available for many of the kinases represented on the Human
Phospho-MAPK Array. Please see the following table for relevant catalog numbers
and links.
|