Survivin (Phospho-Thr34)Colorimetric Cell-Based ELISA Kit (CBCAB01321)

Product Name:	Survivin (Phospho-Thr34)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01321
ELISA Type:	Cell-Based
Target:	Survivin (Phospho-Thr34)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Survivin (Phospho-Thr34) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Survivin protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Survivin in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Survivin phosphorylation.

Qualitative determination of Survivin (Phospho-Thr34) concentration is achieved by an indirect ELISA format. In essence, Survivin (Phospho-Thr34) is captured by Survivin (Phospho-Thr34)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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.

Database Information:	Gene ID: 332, UniProt ID: O15392, OMIM: 603352, Unigene: Hs.514527
Gene Symbol:	BIRC5
Sub Type:	Phospho

UniProt Protein Function:	Survivin: an apoptosis inhibitor that is expressed during the G2/M phase of the cell cycle. Associates with the microtubules of the mitotic spindle and any disruption results in the loss of apoptosis activity. May play a role in neoplasia. Inhibitor of caspase-3 and caspase-7. Two splice variant isoforms have been described.
UniProt Protein Details:	Protein type:Apoptosis Chromosomal Location of Human Ortholog: 17q25 Cellular Component: centriole; nuclear chromosome; microtubule; spindle microtubule; cytoplasmic microtubule; cytoplasm; spindle; midbody; interphase microtubule organizing center; nucleus; cytosol; chromosome, pericentric region Molecular Function:tubulin binding; identical protein binding; protein homodimerization activity; zinc ion binding; microtubule binding; metal ion binding; caspase inhibitor activity; ubiquitin-protein ligase activity; protein binding; enzyme binding; protein heterodimerization activity; chaperone binding; cofactor binding; cysteine protease inhibitor activity; Ran GTPase binding; cobalt ion binding Biological Process: mitosis; transcription, DNA-dependent; spindle checkpoint; establishment of chromosome localization; regulation of signal transduction; protein ubiquitination; cytokinesis; negative regulation of caspase activity; positive regulation of mitotic cell cycle; protein amino acid phosphorylation; chromosome segregation; protein complex localization; cell division; positive regulation of cell proliferation; mitotic cell cycle; G2/M transition of mitotic cell cycle; negative regulation of transcription, DNA-dependent; negative regulation of apoptosis; positive regulation of exit from mitosis
NCBI Summary:	This gene is a member of the inhibitor of apoptosis (IAP) gene family, which encode negative regulatory proteins that prevent apoptotic cell death. IAP family members usually contain multiple baculovirus IAP repeat (BIR) domains, but this gene encodes proteins with only a single BIR domain. The encoded proteins also lack a C-terminus RING finger domain. Gene expression is high during fetal development and in most tumors, yet low in adult tissues. Alternatively spliced transcript variants encoding distinct isoforms have been found for this gene. [provided by RefSeq, Jun 2011]
UniProt Code:	O15392
NCBI GenInfo Identifier:	380865472
NCBI Gene ID:	332
NCBI Accession:	O15392.3
UniProt Secondary Accession:	O15392,Q2I3N8, Q4VGX0, Q53F61, Q5MGC6, Q6FHL2, Q75SP2 Q9P2W8, A2SUH6, B2R4R1,
UniProt Related Accession:	O15392
Molecular Weight:	8,490 Da
NCBI Full Name:	Baculoviral IAP repeat-containing protein 5
NCBI Synonym Full Names:	baculoviral IAP repeat containing 5
NCBI Official Symbol:	BIRC5
NCBI Official Synonym Symbols:	API4; EPR-1
NCBI Protein Information:	baculoviral IAP repeat-containing protein 5; apoptosis inhibitor 4; survivin variant 3 alpha; apoptosis inhibitor survivin
UniProt Protein Name:	Baculoviral IAP repeat-containing protein 5
UniProt Synonym Protein Names:	Apoptosis inhibitor 4; Apoptosis inhibitor survivin
Protein Family:	Baculoviral IAP repeat-containing protein
UniProt Gene Name:	BIRC5
UniProt Entry Name:	BIRC5_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
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-Survivin (Phospho-Thr34) Antibody, Anti-Survivin 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.)