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Product Description
Specifications
| Size | 1 Plate Kit, 2 Plate Kit, 5 Plate Kit |
| Estimated Lead Time | 1-2 business days |
| Species | Human |
| Specificity | The antibody pair provided in this kit recognizes human Phospho-HSP27 (pSer82). |
| Compatible Sample Types | Tissue Lysates, Cell Lysates |
| Accession Number | P04792 |
| Gene Id | 3315 |
| Gene Symbols | HSPB1, HSP27, HSP28 |
| Protein Name / Synonyms | Heat shock protein beta-1 (HspB1) (28 kDa heat shock protein) (Estrogen-regulated 24 kDa protein) (Heat shock 27 kDa protein) (HSP 27) (Stress-responsive protein 27) (SRP27) |
| Quantitative/Semi-Quantitative | Semi-Quantitative |
| Solid Support | 96-well Microplate |
| Design Principle | Sandwich-based |
| Method Of Detection | Colorimetric |
| Research Area | Angiogenesis, Apoptosis, Cancer, Post-Translational Modifications, Phosphorylation, Autophagy, Protein Folding |
| Shipping Type | Blue ice |
| Storage | -20°C |
Introduction
RayBio® Phospho- HSP27 (S82) and Total HSP27 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 HSP27 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-HSP27 and total HSP27. An anti-pan HSP27 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and HSP27 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-HSP27 (S82) antibody is used to detect phosphorylated HSP27 or biotinylated anti-HSP27 antibody is used to detect pan HSP27. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-conjugated Streptavidin 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 HSP27 (S82) or pan HSP27 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human phospho-HSP27 and total HSP27. An anti-pan HSP27 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and HSP27 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-HSP27 (S82) antibody is used to detect phosphorylated HSP27 or biotinylated anti-HSP27 antibody is used to detect pan HSP27. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-conjugated Streptavidin 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 HSP27 (S82) or pan HSP27 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Product Features
- Simultaneously measure phosphorylated protein and pan protein in one experiment (for normalization purpose)
- 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
- Anti-Pan Antibody (*not included in phospho-only version)
- 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
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2.5 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared 1X HRP-Streptavidin to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Typical Data
Positive Control
HeLa cells were treated with PMA at 37°C for 20 min. 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.
PMA Stimulation of HeLa Cell Lines
HeLa cells were treated or untreated with 250 nM PMA for 20 min. Cell lysates were analyzed using this phospho-ELISA 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. 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.