Tubulin gamma Colorimetric Cell-Based ELISA Kit (CBCAB00894)

Product Name:	Tubulin gamma Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00894
ELISA Type:	Cell-Based
Target:	Tubulin gamma
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of Tubulin gamma concentration is achieved by an indirect ELISA format. In essence, Tubulin gamma is captured by Tubulin gamma-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 7283, UniProt ID: P23258, OMIM: 191135, Unigene: Hs.279669
Gene Symbol:	TUBG1
Sub Type:	None

UniProt Protein Function:	TUBG1: Tubulin is the major constituent of microtubules. Gamma tubulin is found at microtubule organizing centers (MTOC) such as the spindle poles or the centrosome. Pericentriolar matrix component that regulates alpha/beta tubulin minus-end nucleation, centrosome duplication and spindle formation. Interacts with GCP2 and GCP3. Interacts with B9D2. Interacts with CDK5RAP2; the interaction is leading to centrosomal localization of TUBG1 and CDK5RAP2. Interacts with PIFO/C1orf88. Belongs to the tubulin family.
UniProt Protein Details:	Protein type:Cytoskeletal; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 17q21 Cellular Component: centriole; centrosome; pericentriolar material; recycling endosome; condensed nuclear chromosome; apical part of cell; cytoplasmic microtubule; nonmotile primary cilium; cytoplasm; leading edge; polar microtubule; cytosol; gamma-tubulin complex Molecular Function:GTPase activity; protein binding; GTP binding; structural constituent of cytoskeleton Biological Process: metabolic process; organelle organization and biogenesis; microtubule cytoskeleton organization and biogenesis; mitotic cell cycle; G2/M transition of mitotic cell cycle; microtubule nucleation; meiotic spindle organization and biogenesis; cytoplasmic microtubule organization and biogenesis Disease: Cortical Dysplasia, Complex, With Other Brain Malformations 4
NCBI Summary:	This gene encodes a member of the tubulin superfamily. The encoded protein localizes to the centrosome where it binds to microtubules as part of a complex referred to as the gamma-tubulin ring complex. The protein mediates microtubule nucleation and is required for microtubule formation and progression of the cell cycle. A pseudogene of this gene is found on chromosome 7. [provided by RefSeq, Jan 2009]
UniProt Code:	P23258
NCBI GenInfo Identifier:	20455518
NCBI Gene ID:	7283
NCBI Accession:	P23258.2
UniProt Secondary Accession:	P23258,Q53X79, Q9BW59,
UniProt Related Accession:	P23258
Molecular Weight:	451
NCBI Full Name:	Tubulin gamma-1 chain
NCBI Synonym Full Names:	tubulin, gamma 1
NCBI Official Symbol:	TUBG1
NCBI Official Synonym Symbols:	TUBG; GCP-1; CDCBM4; TUBGCP1
NCBI Protein Information:	tubulin gamma-1 chain; tubulin, gamma polypeptide; gamma-tubulin complex component 1
UniProt Protein Name:	Tubulin gamma-1 chain
UniProt Synonym Protein Names:	Gamma-1-tubulin; Gamma-tubulin complex component 1; GCP-1
UniProt Gene Name:	TUBG1
UniProt Entry Name:	TBG1_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-Tubulin gamma 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.)