Tubulin Assay Kit

종류 및 특징
• Tubulin Polymerization Assay Kit
  • 고순도의 neuronal tubulin이 키트에 포함되어 있습니다.
  • polymerization curve 결과로 microtubule polymerization, nucleation, equilibrium 세가지 단계를 관찰할 수 있습니다.
  • Tubulin의 순도에 따라 여러가지 그레이드로 제공됩니다.

  Cat. No.	Tubulin	Detection Method
  BK004 & CDS01	>97% pure bovine
  BK006 & CDS03	>99% pure bovine
  BK004P	>97% pure porcine
  BK006P	>99% pure porcine
  BK011	>99% pure bovine	fluorescence based
  BK011P	>99% pure porcine	fluorescence based

  • 제품의 구성:
    • Tubulin
    • GTP solution (Cat. # BST06)
    • General tubulin buffer (PEM, Cat. # BST01)
    • Tubulin glycerol buffer (Cat. # BST05)
    • Paclitaxel positive control (Cat. # TXD01)
    • DMSO for paclitaxel
    • Half area 96-well plate for polymerization reactions
    • Manual with detailed protocols and extensive troubleshooting guide
  • Fluorescence vs Absorbance Assay 차이

    Assay Characteristics	Absorbance Assay	Fluorescence Assay
    Tubulin used per assay	300 μg	100 μg
    Volume of reaction	100 μl	50 μl
    Signal to noise ratio	2	4
    Coefficient of variation (cv)*	13%	11%
    Paclitaxel EC50**	1 μM	1 μM
    Vinblastine IC50 **	0.6 μM	0.6 μM
    Possible problems	Glycerol in standard assay format may interfere with drug or protein binding. Assay conditions can easily be altered to test this.	Fluorescent reporter may interfere with drug or protein binding.

    *: Duplicate samples
    **: Under standard assay conditions. Conditions can be optimized for polymerization enhancers or inhibitors.
  • 적용
    • Screening compounds for effects on tubulin polymerization activity.
    • Screening proteins for effects on tubulin polymerization activity.
    • Determining that tubulin alone (not MAPs) is the target for compounds that affect microtubule polymerization.
    • Teaching aid for undergraduate/graduate class in pharmacology.
• 사용예
  • The BK006P kit was used to study the effects of Paclitaxel, a polymerization enhancer and Nocodazole, a polymerization inhibitor on tubulin polymerization (Fig. 1)
    

    Figure 1. Tubulin polymerization curves from kit BK006P. The figure shows a standard polymerization curve (Control curve) containing a 100 μl volume of 3 mg/ml tubulin in 80 mM PIPES pH 7.0, 0.5 mM EGTA, 2 mM MgCl2, 1 mM GTP and 10% glycerol. Polymerization was started by incubation at 37°C and followed by absorption readings at 340 nm. Under these conditions, polymerization Vmax is enhanced 4 fold in the presence of 10 μM paclitaxel and reduced 5.5 fold in the presence of 10 μM nocodazole.
  • Compounds or proteins that interact with tubulin will often alter one or more of the characteristic phases of polymerization. For example, Figure 2 shows the effect of adding the anti-mitotic drug paclitaxel to the standard polymerization reaction. A 3 μM concentration of paclitaxel eliminates the nucleation phase and enhances the Vmax of the growth phase. Thus, one application of this assay is the identification of novel anti-mitotics. Figure 1 also shows the effect of adding the microtubule destabilizing drug, vinblastine. At 3 μM final concentration, vinblastine causes a drastic decrease in Vmax and reduction in final polymer mass.
    

    Figure 2. Tubulin polymerization using the fluorescence based tubulin polymerization assay (BK011). Tubulin was incubated alone (Control), with Paclitaxel or Vinblastine. Each condition was tested in duplicate. Polymerization was measured by excitation at 360 nm and emission at 420 nm. The three Phases of tubulin polymerization are marked for the control polymerization curve; I: nucleation, II: growth, III: steady state equillibrium.
• Micotubule Binding Protein Spin-down Assay Kit (Cat# BK029)
  • 제품의 구성:
    • Tubulin, >99% pure, (Cat. # TL238)
    • Microtubule associated protein fraction (positive control) (Cat. # MAPF)
    • Bovine serum albumin (BSA) protein (negative control)
    • General tubulin buffer (Cat. # BST01)
    • Tubulin glycerol buffer (Cat. # BST05)
    • GTP (Cat. # BST06)
    • Microtubule resuspension buffer
    • Paclitaxel (Cat. # TXD01)
    • DMSO
    • Salt-extraction buffer
  • 적용
    • To determine whether a protein or compound binds to microtubules.
    • To test various deletion mutants for their ability to still bind microtubules, then calculate their affinity for microtubules, and hence identify the microtubule binding site.
    • In the presence of a non-hydrolyzable analog of ATP (kinesins) or GTP (dynamin) extract these from cells or show their binding affinity when mutated.
• Microtubules/tubulin Biochem Kit (Cat# BK015)
  • 제품의 구성:
    • 8 x 250 μg Tubulin, >99% pure (Cat. # TL238)
    • General Tubulin Buffer (Cat. # BST01)
    • GTP (Cat. # BST06)
    • Microtubule glycerol Buffer (Cat. # BST05)
    • Taxol Stock Solution (Cat. # TXD01)
    • Dry DMSO
  • 적용
    • Test binding activity of known or proteins or compounds to tubulin or microtubules.
    • Test polymerization in the presence of microtubule / tubulin binding proteins.
    • Test conditions that may perturb MAP binding.
  • Product Citation
    • van Horck, F. P., Ahmadian, M. R., Haeusler, L. C., Moolenaar, W. H. and Kranenburg, O. (2001). Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules. J. Biol. Chem. 276, 4948-4956.
• Microtubule / tubulin in vivo Kit (Cat# BK038)
  • 제품의 구성:
    • Lysis and Microtubule stabilization buffer
    • GTP (Cat. # BST06)
    • ATP (Cat. # BSA04)
    • Protease inhibitor cocktail
    • Microtubule enhancing control solution
    • Microtubule depolymerization control solution
    • Control Tubulin Standard (Cat. # TL238)
    • Tubulin monoclonal antibody (Cat. # ATN01)
    • SDS sample buffer (5 x)
    • DMSO
  • 적용
    • To study the effects of pharmaceutical compounds on the ratio of Tubulin to Microtubules in cells.
    • To study the effects of mutated cell lines versus their parent cell line for the change in ratio of Tubulin to Microtubules.
    • To study the effects of physical alterations of environment on the ratio of Tubulin to Microtubules in cells.
  • Product Citation
    • Davis, F. J., Pillai, J. B., Gupta, M. and Gupta, M. P. (2005). Concurrent opposite effects of trichostatin A, an inhibitor of histone deacetylases, on expression of α-MHC and cardiac tubulins: implication for gain in cardiac muscle contractility. Am. J. Physiol. 288, H1477-1490.
• Fluorescent microtubules Biochem Kit (Rhodamine) (Cat# BK007R)
  • 제품의 구성:
    • 1. 5 x 20 μg Rhodamine Labeled Tubulin (Cat. # TL331M).
    • 2. 2 x 250 μg Tubulin Plus Glycerol (Cat. # TL238).
    • 3. General Tubulin Buffer (Cat. # BST01).
    • 4. GTP in PEM Buffer (Cat. # BST06).
    • 5. Tubulin Glycerol Buffer (Cat. # BST05).
    • 6. Antifade
  • 적용
    • Create fluorescent microtubules for in vitro motility assays.
    • Use separate components for separate experiments (e.g. microinjection and biochemical assays).
    • Teaching aid for undergraduate or graduate classes.
  • 사용예
    
    Figure 1. Rhodamine labeled microtubules created with kit BK007R
• Kinesin/dynein motility Kit (Cat# BK027)
  • 제품의 구성:
    • Tubulin, >99% pure, lyophilized (Cat. # TL238)
    • Rhodamine tubulin, lyophilized (Cat. # TL331M)
    • Kinesin heavy chain motor domain, lyophilized (positive control) (Cat. # KR01)
    • Antifade
    • ATP (Cat. # BSA04)
    • Microtubule polymerization buffer
    • Tubulin glycerol buffer
    • Microtubule resuspension buffer
    • Blocking solution
    • Chamber wash buffer
    • Motility buffer
    • Paclitaxel (Cat. # TXD01)
    • DMSO
    • 25 acid washed perfusion chambers
    • Chamber filters
  • 적용
    • To determine whether a protein or compound alters kinesin or dynein-microtubule mobility.
    • To test other proteins for microtubule mobility.
  • 사용예
    
    Figure 1. Microtubule Motility Assay. The field was selected to show a sparse area of MT coverage in order to allow clear visualization of MT motility. Each frame represents a 5 min time-point, from 0 to 15 min. The position of each MT at time zero is marked by an arrow. The arrow position remains identical in each frame to serve as a reference point for MT movement. To obtain an average MT velocity, we recommend measure motility of approximately 30 - 50 individual MTs.