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Tubulin (HTS format, porcine, 8 assays)

Hersteller Cytoskeleton
Kategorie
Typ Proteins
Specific against Porcine
Menge 1 x 4 mg
ArtNr HTS03-A
eClass 6.1 34160400
eClass 9.0 42020190
Lieferbar
Additional info

Product Uses Include

  • Economical alternative to T240 in primary screens for tubulin ligand drugs
  • High-throughput tubulin polymerization or depolymerization screens
  • Production of microtubule substrates for HTS motor assays

Material
Tubulin protein has been isolated from porcine brain. The final product contains approximately 97% tubulin and 3% Microtubule Associated Proteins (MAPs). This version of porcine tubulin is an economical alternative to our highly purified tubulin (Cat. # T240) for anti-tubulin ligand drug discovery. It has been formulated to be compatible with 96 or 384-well format polymerization assays such as in Biochem kits BK004P or BK011P .


Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS-PAGE gel. HTS02 contains 97% tubulin (MW. 55 kDa) and 3% MAPs (MW 35-280 kDa).

Figure 1: A 100 ug sample of HTS03 protein was separated by electrophoresis on a 12% SDS-PAGE gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat.# ADV02).

Biological Activity
An ASSAY UNIT is defined as the amount of HTS03 protein needed to achieve a tubulin polymerization signal of OD340 of 0.1 - 0.15 in 30 minutes at 37C in G-PEM buffer. The assay volume is 100 ul and assumes a spectrophotometer pathlength of 0.5 cm. The protein concentration for HTS02 in this assay is approximately 4 mg/ml. The biological activity of HTS03 is assessed by a tubulin polymerization assay. One ASSAY UNIT of tubulin is used for each polymeriztion assay. An OD340 of 0.10 - 0.15 is required to pass quality control. Tubulin polymerization must also be responsive to polymerization enhancers (paclitaxel) and inhibitors (nocodazole) at 5 uM drug concentration.

Figure 2: Tubulin polymerization in a 96-well format using HTS03. All samples are in duplicates. Each well has a mini-polymerization curve with 20 min on the x-axis and 0.30 OD 340 nm on the y-axis. The Vmax parameter is used to compare inhibitors, the CV is 13% in this format, so a 50% cut off is required for a 98% confidence.

Delivery Time
1-2 Weeks
Shipping Temp.
AT
Storage on Arrival
4C
faqs

 

Question 1:  Does tubulin polymerization using 97% pure porcine brain tubulin (Cat. # HTS03) require additional enhancers such as glycerol or taxol?

Answer 1:  No additional enhancers are required when using 3 or 4 mg/ml of the MAP-enriched 97% pure tubulin.  The recommended polymerization reaction using 97% pure tubulin contains 100 &mu, l of 3 or 4 mg/ml tubulin in 80 mM PIPES pH 6.9, 0.5 mM EGTA, 2 mM MgCl2 and 1 mM GTP.  Polymerization is started by incubation at 37°, C and followed by absorption readings at 340 nm. Under these conditions, polymerization will reach a maximal OD340 between 0.15 &ndash, 0.25 within 30 minutes. In this experimental set up (100 &mu, l volume in a spectrophotometer with a pathlength of 0.5 cm), an OD340 of 0.1 is approximately equal to 1 mg per ml of tubulin polymer mass.  Under these conditions only 40% of the tubulin is polymerized, offering the flexibity to study how polymerization enhancers (e.g., taxol) or inhibitors (e.g., nocodazole) alter tubulin polymerization.  For enhancers, we recommend using 3 mg/ml tubulin whereas for inhibitors, 4 mg/ml tubulin works better.

 

Question 2:  Upon resuspension as directed, what are the components of the final buffer?

Answer 2:  Upon reconstitution as directed, the tubulin will be in the following buffer: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 7.0, 1 mM GTP, 1% Ficoll, 5% sucrose and <, 1mM DTT.

 

 

If you have any questions concerning this product, please contact our Technical Service department at infohoelzel.de

Hinweis: Die dargestellten Informationen und Dokumente (Bedienungsanleitung, Produktdatenblatt, Sicherheitsdatenblatt und Analysezertifikat) entsprechen unserem letzten Update und sollten lediglich der Orientierung dienen. Wir übernehmen keine Garantie für die Aktualität. Für spezifische Anforderungen bitten wir Sie, uns eine Anfrage zu stellen.

Alle Produkte sind nur für Forschungszwecke bestimmt. Nicht für den menschlichen, tierärztlichen oder therapeutischen Gebrauch.

Menge: 1 x 4 mg
Lieferbar: In stock
lieferbar

Lieferung vsl. bis 18.04.2024 

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