RBM5 Colorimetric Cell-Based ELISA

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SKU:
CBCAB01035
€499

Description

Technical Manual

RBM5 Cell-Based ELISA Kit

The RBM5 Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor RBM5 protein expression profile in cells. The kit can be used for measuring the relative amounts of RBM5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on RBM5 .

How does our RBM5 Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of RBM5 concentration is achieved by an indirect ELISA format. In essence, RBM5 is captured by RBM5 -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.

RBM5 Colorimetric Cell-Based ELISA - Information

Product Name:RBM5 Colorimetric Cell-Based ELISA
Product Code:/SKUCBCAB01035
Description:The RBM5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect RBM5 protein expression profile in cells. The kit can be used for measuring the relative amounts of RBM5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on RBM5.
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: 10181, UniProt ID: P52756, OMIM #: 606884, Unigene #: Hs.439480
Format:96-Well Microplate
NCBI Gene Symbol:RBM5
Sub Type:None
Target Name:RBM5

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-RBM5 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.)

RBM5 - Protein Information

UniProt Protein Function:RBM5: Component of the spliceosome A complex. Regulates alternative splicing of a number of mRNAs. May modulate splice site pairing after recruitment of the U1 and U2 snRNPs to the 5' and 3' splice sites of the intron. May both positively and negatively regulate aopotosis by regulating the alternative splicing of several genes involved in this process, including FAS and CASP2/caspase-2. In the case of FAS, promotes exclusion of exon 6 thereby producing a soluble form of FAS that inhibits apoptosis. In the case of CASP2/caspase-2, promotes exclusion of exon 9 thereby producing a catalytically active form of CASP2/Caspase-2 that induces apoptosis. Belongs to the RBM5/RBM10 family. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:RNA splicing; C2H2-type zinc finger protein; Apoptosis; Cell cycle regulation; RNA processing; RNA-binding

Chromosomal Location of Human Ortholog: 3p21.3

Cellular Component: nucleoplasm; nucleus; spliceosome

Molecular Function:DNA binding; mRNA binding; protein binding; RNA binding

Biological Process: negative regulation of cell proliferation; nuclear mRNA splicing, via spliceosome; positive regulation of apoptosis; regulation of alternative nuclear mRNA splicing, via spliceosome; RNA processing; spliceosome assembly

NCBI Summary:This gene is a candidate tumor suppressor gene which encodes a nuclear RNA binding protein that is a component of the spliceosome A complex. The encoded protein plays a role in the induction of cell cycle arrest and apoptosis through pre-mRNA splicing of multiple target genes including the tumor suppressor protein p53. This gene is located within the tumor suppressor region 3p21.3, and may play a role in the inhibition of tumor transformation and progression of several malignancies including lung cancer. [provided by RefSeq, Oct 2011]
UniProt Code:P52756
NCBI GenInfo Identifier:13124794
NCBI Gene ID:10181
NCBI Accession:P52756.2
UniProt Secondary Accession:P52756,Q93021, Q9BU14, Q9HDA6, Q9UKY8, Q9UL24, B2RA45 B4DM16, B4DMF9, B4DZ63,
UniProt Related Accession:P52756
Molecular Weight:17,891 Da
NCBI Full Name:RNA-binding protein 5
NCBI Synonym Full Names:RNA binding motif protein 5
NCBI Official Symbol:RBM5  
NCBI Official Synonym Symbols:G15; H37; RMB5; LUCA15  
NCBI Protein Information:RNA-binding protein 5
UniProt Protein Name:RNA-binding protein 5
UniProt Synonym Protein Names:Protein G15; Putative tumor suppressor LUCA15; RNA-binding motif protein 5; Renal carcinoma antigen NY-REN-9
Protein Family:RNA-binding protein
UniProt Gene Name:RBM5  
UniProt Entry Name:RBM5_HUMAN
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