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• Can other lysis buffers be used?
• Can a non-film based image acquisition system, such as a cooled CCD camera, be used?
• Can the array be stripped and reused?
• What exposure times should be used for chemiluminescent detection with X-ray film?
• Can the sensitivity be increased by altering the Detection Antibody Cocktail or Streptavidin-HRP concentration?
• What is the sensitivity of the Phospho-MAPK Array?
• Can tissue lysates be used?
• How is it known that the spotted capture antibodies are actually working? What samples were used for testing?
• 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?
• 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?

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.

Kinase	DuoSet IC Catalog #	Surveyor IC Catalog #
ERK1	DYC1825
ERK2	DYC1483	SUV1483
JNK2	DYC2236
JNK pan	DYC1387
p38γ	DYC1664
p38δ	DYC2124
p38α	DYC869	SUV869
RSK1	DYC892
Akt1	DYC2289
Akt pan	DYC887	SUV887
GSK-3 α/β	DYC2630
HSP27	DYC2314
p70 S6 Kinase	DYC8965