Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total α-Tubulin Sandwich ELISA Kit #7944

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
Alpha-Tubulin Rabbit Antibody Coated Microwells 96 tests
Alpha-Tubulin Mouse Detection Ab 11 ml Green
Anti-mouse IgG, HRP- linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M R Hm Mk

Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Total α-Tubulin Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of α-tubulin. An α-tubulin rabbit antibody has been coated onto the microwells. After incubation with cell lysates, α-tubulin is captured by the coated antibody. Following extensive washing, an α-tubulin mouse detection antibody is added to detect the captured α-tubulin. An anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate (TMB) is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of α-tubulin.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total α-Tubulin Sandwich ELISA Kit detects endogenous levels of α-tubulin protein, as shown in Figure 1. The kit sensitivity is shown in Figure 2. Microtubule stabilizing or destabilizing agents may significantly increase or decrease the signal, respectively. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of A-431 cells with EGF stimulates phosphorylation of the EGF receptor at Tyr1086 as detected by PathScan® Phospho-EGF Receptor (Tyr1086) Sandwich ELISA Kit #7240, but does not affect the level of α-tubulin as detected by PathScan® Total α-Tubulin Sandwich ELISA Kit #7944. A-431 cells were treated with EGF (100 ng/ml, 3 min.) at 37ºC before lysis. Absorbance at 450 nm is shown in the top figure, while the corresponding western blots using Total α-Tubulin (11H10) Rabbit mAb #2125 (left) or Phospho-EGF Receptor (Tyr1086) Antibody #2220 (right) are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of the lysate from A-431 cells and the absorbance at 450 nm is shown.

Background

The cytoskeleton consists of three types of cytosolic fibers: microtubules, microfilaments (actin filaments), and intermediate filaments. Globular tubulin subunits comprise the microtubule building block, with α/β-tubulin heterodimers forming the tubulin subunit common to all eukaryotic cells. γ-tubulin is required to nucleate polymerization of tubulin subunits to form microtubule polymers. Many cell movements are mediated by microtubule action, including the beating of cilia and flagella, cytoplasmic transport of membrane vesicles, chromosome alignment during meiosis/mitosis, and nerve-cell axon migration. These movements result from competitive microtubule polymerization and depolymerization or through the actions of microtubule motor proteins (1).

  1. Westermann, S. and Weber, K. (2003) Nat. Rev. Mol. Cell Biol. 4, 938 -947.

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