Stathmin 1 (Phospho-Ser24) Cell-Based ELISA Kit

Product Name:	Stathmin 1 (Phospho-Ser24)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01612
ELISA Type:	Cell-Based
Target:	Stathmin 1 (Phospho-Ser24)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Stathmin 1 (Phospho-Ser24) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Stathmin 1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Stathmin 1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Stathmin 1 phosphorylation.

Qualitative determination of Stathmin 1 (Phospho-Ser24) concentration is achieved by an indirect ELISA format. In essence, Stathmin 1 (Phospho-Ser24) is captured by Stathmin 1 (Phospho-Ser24)-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: 3925, UniProt ID: P16949, OMIM: 151442, Unigene: Hs.209983
Gene Symbol:	STMN1
Sub Type:	Phospho

UniProt Protein Function:	STMN1: a microtubule-associated protein involved in the regulation of the microtubule filament system by destabilizing microtubules. It prevents assembly and promotes disassembly of microtubules. Phosphorylation reduces tubulin binding 10-fold and suppresses the MT polymerization inhibition activity. Present in much greater abundance in cells from patients with acute leukemia of different subtypes than in normal peripheral blood lymphocytes, nonleukemic proliferating lymphoid cells, bone marrow cells, or cells from patients with chronic lymphoid or myeloid leukemia.
UniProt Protein Details:	Protein type:Cytoskeletal Chromosomal Location of Human Ortholog: 1p36.11 Cellular Component: cytoplasm; cytosol; intracellular; membrane; microtubule; neuron projection Molecular Function:protein binding; signal transducer activity; tubulin binding Biological Process: axonogenesis; brain development; microtubule depolymerization; mitotic spindle organization and biogenesis; negative regulation of microtubule polymerization; neurite development; positive regulation of cell motility; regulation of cytoskeleton organization and biogenesis; response to virus; signal transduction
NCBI Summary:	This gene belongs to the stathmin family of genes. It encodes a ubiquitous cytosolic phosphoprotein proposed to function as an intracellular relay integrating regulatory signals of the cellular environment. The encoded protein is involved in the regulation of the microtubule filament system by destabilizing microtubules. It prevents assembly and promotes disassembly of microtubules. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Feb 2009]
UniProt Code:	P16949
NCBI GenInfo Identifier:	134973
NCBI Gene ID:	3925
NCBI Accession:	P16949.3
UniProt Secondary Accession:	P16949,A2A2D1, B2R4E7, B7Z8N4, D3DPJ5,
UniProt Related Accession:	P16949
Molecular Weight:	19,824 Da
NCBI Full Name:	Stathmin
NCBI Synonym Full Names:	stathmin 1
NCBI Official Symbol:	STMN1
NCBI Official Synonym Symbols:	Lag; SMN; OP18; PP17; PP19; PR22; LAP18; C1orf215
NCBI Protein Information:	stathmin
UniProt Protein Name:	Stathmin
UniProt Synonym Protein Names:	Leukemia-associated phosphoprotein p18; Metablastin; Oncoprotein 18; Op18; Phosphoprotein p19; pp19; Prosolin; Protein Pr22; pp17
Protein Family:	Stathmin
UniProt Gene Name:	STMN1
UniProt Entry Name:	STMN1_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-Stathmin 1 (Phospho-Ser24) Antibody, Anti-Stathmin 1 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.)