MED26 Colorimetric Cell-Based ELISA

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SKU:
CBCAB00962
€499

Description

Technical Manual

MED26 Cell-Based ELISA Kit

The MED26 Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor MED26 protein expression profile in cells. The kit can be used for measuring the relative amounts of MED26 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on MED26 .

How does our MED26 Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of MED26 concentration is achieved by an indirect ELISA format. In essence, MED26 is captured by MED26 -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

MED26 Colorimetric Cell-Based ELISA - Information

Product Name:MED26 Colorimetric Cell-Based ELISA
Product Code:/SKUCBCAB00962
Description:The MED26 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MED26 protein expression profile in cells. The kit can be used for measuring the relative amounts of MED26 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MED26.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse
Assay Type:Cell-Based
Database Links:Gene ID: 9441, UniProt ID: O95402, OMIM #: 605043, Unigene #: Hs.651587
Format:96-Well Microplate
NCBI Gene Symbol:MED26
Sub Type:None
Target Name:MED26

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 ml
Quenching Buffer24 ml
Blocking Buffer50 ml
15X Wash Buffer50 ml
Primary Antibody Diluent12 ml
100x Anti-Phospho Target Antibody 60 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
HRP-Conjugated Anti-Rabbit IgG Antibody12 ml
HRP-Conjugated Anti-Mouse IgG Antibody12 ml
SDS Solution12 ml
Stop Solution24 ml
Ready-to-Use Substrate12 ml
Crystal Violet Solution12 ml
Adhesive Plate Seals2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µl to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.Seed 200 µl of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5.Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µl of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µl of 1x primary antibodies (Anti-MED26 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µl of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µl of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µl of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

MED26 - Protein Information

UniProt Protein Function:MED26: Component of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Mediator functions as a bridge to convey information from gene-specific regulatory proteins to the basal RNA polymerase II transcription machinery. Mediator is recruited to promoters by direct interactions with regulatory proteins and serves as a scaffold for the assembly of a functional preinitiation complex with RNA polymerase II and the general transcription factors. Belongs to the Mediator complex subunit 26 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Transcription, coactivator/corepressor

Chromosomal Location of Human Ortholog: 19p13.11

Cellular Component: nucleoplasm; Srb-mediator complex

Molecular Function:protein binding; transcription coactivator activity

Biological Process: regulation of transcription from RNA polymerase II promoter; transcription initiation from RNA polymerase II promoter

NCBI Summary:The activation of gene transcription is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus. The protein encoded by this gene is a subunit of the CRSP (cofactor required for SP1 activation) complex, which, along with TFIID, is required for efficient activation by SP1. This protein is also a component of other multisubunit complexes e.g. thyroid hormone receptor-(TR-) associated proteins which interact with TR and facilitate TR function on DNA templates in conjunction with initiation factors and cofactors. [provided by RefSeq, Jul 2008]
UniProt Code:O95402
NCBI GenInfo Identifier:158931130
NCBI Gene ID:9441
NCBI Accession:O95402.2
UniProt Secondary Accession:O95402,Q0VGB6, A1A4S3,
UniProt Related Accession:O95402
Molecular Weight:10,359 Da
NCBI Full Name:Mediator of RNA polymerase II transcription subunit 26
NCBI Synonym Full Names:mediator complex subunit 26
NCBI Official Symbol:MED26  
NCBI Official Synonym Symbols:CRSP7; CRSP70  
NCBI Protein Information:mediator of RNA polymerase II transcription subunit 26
UniProt Protein Name:Mediator of RNA polymerase II transcription subunit 26
UniProt Synonym Protein Names:Activator-recruited cofactor 70 kDa component; ARC70; Cofactor required for Sp1 transcriptional activation subunit 7; CRSP complex subunit 7; Mediator complex subunit 26; Transcriptional coactivator CRSP70
Protein Family:Mediator of RNA polymerase II transcription
UniProt Gene Name:MED26  
UniProt Entry Name:MED26_HUMAN
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