Human Phospho-RTK Array Kit FAQs - R&D Systems

Can other lysis buffers be used?

The kit has been validated for use with NP-40 Lysis Buffer.
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 membranes be stripped and reused?

No. Arrays are validated for single-use only.

What exposure times should be used for chemiluminescent detection with X-ray film?

Receptor Tyrosine Kinases (RTK) are expressed at different densities in different cell types. Their level of tyrosine phosphorylation may vary with treatments, length of treatments and concentration of RTK-specific ligand used. Multiple exposures should be obtained in order to acquire as much information as possible from each array experiment. We recommend exposing for 1 to 10 minutes.

Can the sensitivity be increased by altering the conjugate concentration?

Use the recommended concentration of anti-Phospho-Tyrosine-HRP Detection Antibody. Using a higher concentration of detection antibody may result in high background. Longer exposures to X-ray film (5 to 10 minutes) should increase sensitivity.

What is the sensitivity of the Phospho-RTK Array?

Up to 20 times more sensitive than an immunoprecipitation 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 ligand-treated cell cultures or tyrosine phosphorylated recombinant RTKs. Quality Control testing is done on each lot using a panel of lysates prepared from ligand-treated cell cultures.

Can the Phospho-RTK Array be used for measuring the relative levels of total RTKs present in a sample?

The capture antibodies on the array capture both phosphorylated and unphosphorylated RTKs. Lysates from pervanadate-treated cells can be used to measure the relative levels of total RTKs present in a cell line. Pervanadate treatment inactivates phosphatases, allowing phosphorylated tyrosines on RTKs to persist.

I would like to confirm a positive signal for an RTK 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-RTK Array are proprietary information. However, we do sell antibodies for each RTK on the array that are validated for Western blot applications. The anti-phospho-tyrosine-HRP detection antibody is also available (Catalog # HAM1676).