Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Acetyl-α-Tubulin Sandwich ELISA Kit #7204

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

Kit Includes Volume Solution Color
Tubulin Ab-Coated Microwells 96 tests
Acetylated-Lysine Detection Ab 11 ml Green
Anti-rabbit 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 Mk

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

Description

CST's PathScan® Acetyl-α-Tubulin Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetylated α-tubulin protein. An α-tubulin mouse mAb has been coated onto the microwells. After incubation with cell lysates, α-tubulin protein (acetylated and non-acetylated) is captured by the coated antibody. Following extensive washing, an acetyl-lysine rabbit Ab is added to detect the acetylated α-tubulin protein. Anti-rabbit IgG, HRP-linked Antibody #7074 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 acetylated α-tubulin protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Acetyl-α-Tubulin Sandwich ELISA Kit #7204 detects endogenous levels of acetylated α-tubulin. As shown in Figure 1 using the Acetyl-α-Tubulin Sandwich ELISA Kit #7204, a high level of acetylated α-tubulin is detected in COS cells when treated with TSA. The level of total α-tubulin (acetylated and non-acetylated) remains unchanged as shown by Western analysis (Figure 1). Similar results are obtained when NIH/3T3 and Jurkat cells are treated with TSA (data not shown). 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 COS cells with Trichostatin A (TSA) increases the acetylation of α-tubulin detected by PathScan® Acetyl-alpha-Tubulin Sandwich ELISA Kit #7204. TSA treatment does not affect the level of α-tubulin that is detected by Western analysis. COS cells (70-80% confluent) were treated for 16-18 hours with 0.4 micromolar TSA at 37oC. Absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots using α-Tubulin Antibody #2125 (left panel) or Acetylated-Lysine Antibody #9441 (right panel) are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of lysates from untreated and TSA-treated COS cells (see the legend of Figure 1) and the absorbance at 450 nm.

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.

Application References

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