Human Phospho-c-Abl (Y245) and Total c-Abl ELISA

RayBio® Human Phospho-c-Abl (Tyr245) and Total c-Abl ELISA Kit. This assay semi-quantitatively measures c-Abl phosphorylated at Tyrosine-245 as well as total c-Abl in lysate samples.

$541.00  
PEL-ABL1-Y245-T-1
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Antigen Information

Accession Number:
  • P00519
Gene ID:
  • 25
Protein Name & Synonyms:
Tyrosine-protein kinase ABL1 (EC 2.7.10.2) (Abelson murine leukemia viral oncogene homolog 1) (Abelson tyrosine-protein kinase 1) (Proto-oncogene c-Abl) (p150)

Assay Format

Pathway:
  • Cell Cycle
  • DNA Damage
  • HER/ErbB Signaling
Specificity:
The antibody pair provided in this kit recognizes Human c-Abl phosphorylated at site Tyrosine-245 as well as total c-Abl
Number of Targets Detected:
2
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Tissue Lysates
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
  • Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Product Features

  • Rapidly measure phosphorylated protein in lysates
  • Screen numerous different cell lysates without performing a Western Blot analysis
  • Minimal hands-on time, convenient, and non-radioactive material

Application Notes

Kit Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Anti-Phospho Antibody
  • HRP-Conjugated Secondary Antibody
  • Assay Diluent
  • TMB One-Step Substrate
  • Stop Solution
  • Lysis Buffer
  • Positive Control Sample
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Protease and Phosphatase inhibitors
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Benchtop rocker or shaker
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared 1X HRP-Streptavidin to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Positive Control
Jurkat cells were treated with Pervanadate. Solubilize cells at 4 x 10^7 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.
Pervanadate Stimulation of Jurkat Cell Line
Jurkat cells were treated with Pervanadate. Cell lysates were analyzed using this phosphoELISA and Western Blot.


Storage/Stability

Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment.
We ordered Human Phospho-c-Abl (Y245) and Total c-Abl ELISA, 2 plates.
Each kit comes with an AMAZON gift card of $5.00 with an attached note saying you can get a $10 Amazon gift card via email if you review this product.
It is my pleasure to review this product and due to the fact that this review is detailed and that we received 2 kits, I do expect 2 * $10 gift card.
After testing our 36 samples (one replicate) on the 1st plate, we decided we wanted to test only the Total c-Abl on the next set of samples.
Even though the Assay Procedure "strongly recommends to run all positive controls and samples in at least duplicate", the kit only provides enough positive controls for one replicate.
You cannot possibly run more than 1/2 of the plate whether total or phospho since you only get enough primary antibody for 1/2 the plate.
It is unfortunate we receive the standards and both primary lyophilized which doesn't allow to save a concentrate for another experiment.
It is not indicated whether you can freeze/thaw once any of the component has been thawed. Plate, standard etc. The recommendation "Avoid freeze-thaw cycles" is too vague.
The wash concentrate 20X supplied is just enough to wash the plate as the procedure recommends. If you happen to wash by hand, it is certainly not enough.

If you follow the protocol: "bringing all reagents to room temperature" makes no sense if you decide to incubate standards and samples overnight.
I would not recommend "Preparing all reagents prior to starting the assay" especially when you run the test over 2 days.
Overall the test works, but the User Manual is not good.
For the cost of the kit, I can expect more generosity (more standard to match the recommendations, more wash concentrate, and more primary) ​which would give us more flexibility.

University of Michigan
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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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