Human Phospho-SMAD5 (SER463/465) PharmaGenie ELISA Kit (SBRS1974)

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SKU:
SBRS1974
Product Type:
ELISA Kit
Reactivity:
Human
Applications:
ELISA
ELISA Type:
Sandwich

Description

Human Phospho-SMAD5 (SER463/465) PharmaGenie ELISA Kit (SBRS1974)

R Assay Genie Phospho-SMAD5 (Ser463/465) ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human cell lysates. By determining phosphorylated SMAD5 protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis. This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human phospho-SMAD5. An anti-pan SMAD5 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and SMAD5 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-SMAD5 (Ser463/465) antibody is used to detect phosphorylated SMAD5. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of SMAD5 (Ser463/465) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product SKU:SBRS1974
Size:96T
Application:The antibody pair provided in this kit recognizes Human SMAD5 phosphorylated at site Serine-463/465.
Uniprot:Q99717
Gene ID:4090
Gene Names:MADH5 / SMAD5
Pathway:Stem Cell / TGF-beta Signaling
Synonyms:Mothers against decapentaplegic homolog 5 (MAD homolog 5) (Mothers against DPP homolog 5) (JV5-1) (SMAD family member 5) (SMAD 5) (Smad5) (hSmad5)
Target Species:Human
Compatible Sample Types:Cell Lysates, Tissue Lysates
Design Principle:Sandwich-based
Method of Detection:Colorimetric
Quantitative/Semi-Quantitative:Semi-Quantitative
Storage/Stability:Upon receipt, the kit should be stored at -20°C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4°C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge, and store at -20°C. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.
  1. Pre-Coated 96-well Strip Microplate
  2. Wash Buffer
  3. Anti-Phospho Antibody
  4. HRP-Conjugated Secondary Antibody
  5. Assay Diluent
  6. TMB One-Step Substrate
  7. Stop Solution
  8. Lysis Buffer
  9. Positive Control Sample

Other materials and equipment required:

The Assay Genie Human Phospho-SMAD5 (SER463/465) PharmaGenie ELISA Kit (SBRS1974) will require other equipment and materials to carry out the assay. Please see list below for further details.

  • 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 ul 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
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 ul of sample or positive control to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 ul of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 ul of prepared 1X HRP-Streptavidin to each well.
  7. Incubate 1 h at RT.
  8. Add 100 ul of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 ul of Stop Solution to each well.
  11. Read at 450 nm immediately.
Product ImageJurkat cells were treated or untreated with CPT. Cell lysates were analyzed using this phosphoELISA and Western Blot.
Product ImageHeLa cells were treated with BMP2. Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA.
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