Human Phospho-RB (T826) and Total RB ELISA

RayBio® Human Phospho-RB (Thr826) and Total RB ELISA Kit. This assay semi-quantitatively measures RB phosphorylated at Threonine-826 as well as total RB in lysate samples.

$973.80  
PEL-RB-T826-T-2
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Antigen Information

Accession Number:
  • P06400
Gene ID:
  • 5925
Gene Symbols:
  • RB1
Protein Name & Synonyms:
Retinoblastoma-associated protein (p105-Rb) (pRb) (Rb) (pp110)
Target Species:
  • Human

Assay Format

Specificity:
This ELISA kit recognizes Human RB phosphorylated at site Threonine-826 as well as total RB.

Product Specifications

Size:
1, 2, or 5 x 96-Well 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
HT29 cells were treated with Thymidine and Nocodazole. Solubilize cells at 4 x 107 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.
Mitosis Block of HT29 Cell Line
HT29 cells were treated with Thymidine-Nocodazole Block. 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.

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Usage

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

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