CREB Pathway Assay Pathway Assay Kit

Product Discontinued

CREB Pathway Assay Pathway Assay Kit Summary

• Description: This ELISA product range provides a quick, easy, more sensitive, and qualitatively better alternative to western blotting for kinase assay readouts, and it is designed to demonstrate the qualitative differences in the amounts of specific target proteins between samples.
• Specificity: Kit contains (1) 96 well strip plate containing CREB (pS133) 6 pairs of strips (12 strips total) with a kit assay control for the qualitative detection of phosphorylated CREB in tissue lysates, cell lysates and recombinant protein preparations.
• Kit Type: Pathway Assay Kit

Applications/Dilutions

• Dilutions:
  • Immunoassay

Packaging, Storage & Formulations

• Storage: Store at 4C. Do not freeze.

Notes

You may also Mix-and-Match you own Pathway Assay Kit by purchasing catalog number NBP1-71668. Choose up to 6 different targets from this list: Active Beta Catenin (DP S33/S37/T41), AKT1 (pS473), AKT2 (pS474), CREB (pS133), ERK1/2 (pT202/Y204; pT185/Y187), GSK3 alpha (pS21), GSK3 beta (pS9), p38 (pT180/Y182).

View All Available Pathway Assay Kits

Background

Multi-kinase arrays are an alternative analysis platform to traditional Western blots. Novus' Pathway Assay Kits allow for the simultaneous detection of activation states of multiple kinases in a signal pathway on a single multiwell platform. These kits are easier and faster than traditional Western blots (2.5 hour protocol), demonstrate a significantly greater dynamic range and sensitivity, operate with much less sample (5-20 micrograms total protein), and are more cost-effective with up to six data points per assay.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.

FAQs for CREB Pathway Assay Kit (NBP1-71673). (Showing 1 - 3 of 3 FAQ).

1. Is this kit suitable for assays on mouse tissue?
   • I can confirm that our product CREB Pathway Assay [p Ser133] Kit (NBP1-71673) is suitable for mouse, as well as human and rat. It is for tissue lysates, cell lysates and recombinant protein preparations.
2. I am interested in your CREB pathway assay (p Ser133) Kit. I would be examining the tissue samples. Can you tell me how I prepare the sample material for this best?
   • Sonication works well to break up the initial tissue and cells; some people use mini-pestles. So far customers have used brain tissue, kidney and liver tissue successfully and I would think you could use almost any kind of tissue. Heart and muscle likely will need to be first ground in liquid nitrogen or sheared mechanically prior to being added into lysis buffer because they are just tougher tissues. The key for this assay is to use the urea-based 'Denaturing Cell Lysis Buffer' on the protocol page. This ensures a good strong denatured protein product. That is why it was developed, because other extraction buffers such as RIPA and SDS not only interfered with the assay because of their detergent content but even when diluted out did not provide as consistent signals as our recommended buffer. The amount of buffer to use will depend upon the amount of tissue but a good starting point is 5mg wet weight of tissue in 300uL of buffer. After sonication or disruption the goal is a relatively clear solution with no big pieces and not thick like syrup. If there are pieces and/or if it is thick then you'll have to add more lysis buffer. One should also probably spin the sample for 5-10 minutes at 10K to pellet any remaining small pieces and use the cleared lysate for best results. One point to note about using tissue target specific ELISAs is that most likely the target cell that you are wanting to measure the CREB in is only one part of many, many other cell types all of which will have CREB in them, so with tissue, one usually has to use a great deal more total protein extract than if using a synchronized cell population from a plate to get an appropriate signal to noise. Typically customers using tissue extracts have to only dilute the lysis buffer with assay buffer 1:3 to 1:5 (rather than 1:10-1:20) simply because the amount of true target protein from the one or two cell types wanted is in very short supply because of the nature of tissue being a complete mixture of cell types. Thus, you may have to purchase a full plate worth of strips to optimize your extraction conditions first before you start the assay.
3. I would like to know if this assay is suitable for mouse tissue lysates, and if so, is there a specific protocol?
   • Yes, NBP1-71673 is suitable for mouse, and for tissue lysates. The advice our lab gives regarding preparing tissue samples (in general) for use with this kit is as follows: Sonication works well to break up the initial tissue and cells; some people use mini-pestles. So far customers have used brain tissue, kidney and liver tissue successfully and I would think you could use almost any kind of tissue. Heart and muscle likely will need to be first ground in liquid nitrogen or sheared mechanically prior to being added into lysis buffer because they are just tougher tissues. The key for this assay is to use the urea-based 'Denaturing Cell Lysis Buffer' on the protocol page, and also available to purchase ready-made NBP2-22129. This ensures a good denatured protein product. That is why it was developed, because other extraction buffers such as RIPA and SDS not only interfered with the assay because of their detergent content but even when diluted out did not provide as consistent signals as our recommended buffer. The amount of buffer to use will depend upon the amount of tissue but a good starting point is 5 mg wet weight of tissue in 300 uL of buffer. After sonication or disruption the goal is a relatively clear solution with no big pieces and not thick like syrup. If there are pieces and/or if it is thick then you'll have to add more lysis buffer. One should also probably spin the sample for 5-10 minutes at 10K to pellet any remaining small pieces and use the cleared lysate for best results. One point to note is that most likely the target cell that you are wanting to measure the CREB in is only one part of many, many other cell types all of which will have CREB in them, so with tissue, one usually has to use a great deal more total protein extract than if using a synchronized cell population from a plate to get an appropriate signal to noise. Typically customers using tissue extracts have to only dilute the lysis buffer with assay buffer 1:3 to 1:5 (rather than 1:10-1:20) simply because the amount of true target protein from the one or two cell types wanted is in very short supply because of the nature of tissue being a complete mixture of cell types. Thus, you may have to purchase a full plate worth of strips to optimize your extraction conditions f