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TUBB ELISA Kit

TUBB Reactivity: Human Colorimetric Sandwich ELISA 1.56-100 ng/mL Tissue Homogenate
Catalog No. ABIN454567
  • Target See all TUBB ELISA Kits
    TUBB (Tubulin, beta (TUBB))
    Reactivity
    • 3
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Human
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Detection Range
    1.56-100 ng/mL
    Minimum Detection Limit
    1.56 ng/mL
    Application
    ELISA
    Purpose
    This immunoassay kit allows for the in vitro quantitative determination of human beta-tubulin concentrations in tissue homogenates and other biological fluids.
    Sample Type
    Tissue Homogenate
    Analytical Method
    Quantitative
    Specificity
    This assay recognizes recombinant and natural human beta-tubulin.
    Cross-Reactivity (Details)
    No significant cross-reactivity or interference was observed.
    Sensitivity
    < 1 ng/mL
    The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
    Characteristics
    Homo sapiens,Human,Tubulin beta chain,Tubulin beta-5 chain,TUBB,TUBB5,OK/SW-cl.56
    Components
    Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay DiluentB 1 x 10ml Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)
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  • Sample Volume
    100 μL
    Plate
    Pre-coated
    Protocol
    The microtiter plate provided in this kit has been pre-coated with an antibody specific to beta-tubulin. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for beta-tubulin and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain beta-tubulin, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of beta-tubulin in the samples is then determined by comparing the O.D. of the samples to the standard curve.
    Reagent Preparation

    Bring all reagents to room temperature before use. 3 Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 100 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (100 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

    Sample Collection
    Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20 °C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20 °C. other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles. Note: Serum, plasma, and cell culture supernatant samples to be used within 7 days may be stored at 2-8C, otherwise samples must stored at -20 °C (≤ 1 months) or -80 °C (≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. It is recommended that all samples be assayed in duplicate.
    Assay Procedure

    Allow all reagents to reach room temperature. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Arrange and label required number of strips. Prepare all reagents, working standards and samples as directed in the previous sections.
    1. Add 100 uL of Standard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
    2. Remove the liquid of each well, don’t wash.
    3. Add 100 uL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37°C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
    4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    5. Add 100 uL of Detection Reagent B working solution to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
    6. Repeat the aspiration/wash as in step
    4. 7. Add 90 uL of Substrate Solution to each well. Incubate within 30 minutes at 37°C. Protect from light.
    8. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
    9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
    Important Note:
    1. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once.
    2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    3. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
    4. Duplication of all standards and specimens, although not required, is recommended.
    5. When mixing or reconstituting protein solutions, always avoid foaming.
    6. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
    7. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
    8. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.

    Calculation of Results

    Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the beta-tubulin concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

    Restrictions
    For Research Use only
  • Handling Advice
    1. The kit should not be used beyond the expiration date on the kit label.
    2. Do not mix or substitute reagents with those from other lots or sources.
    3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
    4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
    Storage
    4 °C/-20 °C
    Storage Comment
    The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.
  • Target See all TUBB ELISA Kits
    TUBB (Tubulin, beta (TUBB))
    Alternative Name
    TUBB (TUBB Products)
    Synonyms
    M40 ELISA Kit, OK/SW-cl.56 ELISA Kit, TUBB1 ELISA Kit, TUBB5 ELISA Kit, XLOT ELISA Kit, m40 ELISA Kit, ok/sw-cl.56 ELISA Kit, tubb1 ELISA Kit, tubb5 ELISA Kit, TUBB2A ELISA Kit, TUBB2B ELISA Kit, TUBB ELISA Kit, 1t ELISA Kit, B1t ELISA Kit, BETA 56D ELISA Kit, CG9277 ELISA Kit, DTB2 ELISA Kit, Dmbeta1 ELISA Kit, Dmel\\CG9277 ELISA Kit, T ELISA Kit, Tub ELISA Kit, Tubulin ELISA Kit, beta ELISA Kit, beta-Tub ELISA Kit, beta-Tub56D ELISA Kit, beta-tub ELISA Kit, beta-tubulin56D ELISA Kit, beta1 ELISA Kit, beta1-Tubulin ELISA Kit, beta1-tub ELISA Kit, beta1Tub ELISA Kit, beta1t ELISA Kit, beta1tub ELISA Kit, beta56D ELISA Kit, betaTub ELISA Kit, betaTub1 ELISA Kit, beta[[1]] tubulin ELISA Kit, beta[[1]]-tubulin ELISA Kit, betatub(56D) ELISA Kit, 143391_i_at ELISA Kit, 3t ELISA Kit, B3t ELISA Kit, BETA 60D ELISA Kit, CG3401 ELISA Kit, D.m.BETA-60D ELISA Kit, DTB3 ELISA Kit, Dmbeta3 ELISA Kit, Dmel\\CG3401 ELISA Kit, Tub60D ELISA Kit, beta-Tub60D ELISA Kit, beta-Tub6D ELISA Kit, beta3 ELISA Kit, beta3 TU ELISA Kit, beta3-Tub ELISA Kit, beta3-tubulin ELISA Kit, beta3TUB ELISA Kit, beta3Tub ELISA Kit, beta3t ELISA Kit, beta60C ELISA Kit, betaTub3 ELISA Kit, betaTub60C ELISA Kit, beta[[3]] tubulin ELISA Kit, beta[[3]]-Tub ELISA Kit, beta[[3]]-tubulin ELISA Kit, betatub60D ELISA Kit, p50 ELISA Kit, p50/tubulin ELISA Kit, p53 ELISA Kit, p53/tubulin ELISA Kit, 2t ELISA Kit, B2t ELISA Kit, BETA 85D ELISA Kit, BETA2 ELISA Kit, CG9359 ELISA Kit, D.m.BETA-85D ELISA Kit, DTB4 ELISA Kit, Dmbeta2 ELISA Kit, Dmel\\CG9359 ELISA Kit, beta(2)Tu ELISA Kit, beta(2)Tub ELISA Kit, beta-Tub85D ELISA Kit, beta-tub85D ELISA Kit, beta2 ELISA Kit, beta2-tub ELISA Kit, beta2t ELISA Kit, beta2tub ELISA Kit, beta85D ELISA Kit, betaTub2 ELISA Kit, beta[[2]]-tubulin ELISA Kit, ms(3)KK[D] ELISA Kit, 4t ELISA Kit, B4t ELISA Kit, BETA 98B ELISA Kit, CG4869 ELISA Kit, DTB1 ELISA Kit, Dmbeta4 ELISA Kit, Dmel\\CG4869 ELISA Kit, beta-Tub97EF ELISA Kit, beta4 ELISA Kit, beta4t ELISA Kit, beta97F ELISA Kit, betaTub4 ELISA Kit, betaTub98BC ELISA Kit, betaTub98C ELISA Kit, Tub1 ELISA Kit, bmtub1 ELISA Kit, Tub3 ELISA Kit, bmtub3 ELISA Kit, Tub2 ELISA Kit, bmtub2 ELISA Kit, Tub4 ELISA Kit, bmtub4 ELISA Kit, LOC100101153 ELISA Kit, tubulin beta class I ELISA Kit, putative cobalt-zinc-cadmium outer membrane resistance protein ELISA Kit, Tubulin beta chain ELISA Kit, tubulin beta-2A chain ELISA Kit, beta-Tubulin at 56D ELISA Kit, beta-Tubulin at 60D ELISA Kit, beta-Tubulin at 85D ELISA Kit, beta-Tubulin at 97EF ELISA Kit, beta-tubulin ELISA Kit, tubulin beta 6 class V ELISA Kit, beta tubulin ELISA Kit, tubulin beta class I L homeolog ELISA Kit, tubulin, beta 5 class I ELISA Kit, TUBB ELISA Kit, czcC ELISA Kit, tbb-6 ELISA Kit, tubb ELISA Kit, LOC462399 ELISA Kit, betaTub56D ELISA Kit, betaTub60D ELISA Kit, betaTub85D ELISA Kit, betaTub97EF ELISA Kit, Tub1 ELISA Kit, Tub3 ELISA Kit, Tub2 ELISA Kit, Tub4 ELISA Kit, TVAG_525430 ELISA Kit, TVAG_062880 ELISA Kit, TVAG_456920 ELISA Kit, LOC100101153 ELISA Kit, TUBB6 ELISA Kit, Tb927.1.2330 ELISA Kit, Tb927.1.2350 ELISA Kit, Tb927.1.2370 ELISA Kit, Tb927.1.2390 ELISA Kit, tubb.L ELISA Kit, Tubb ELISA Kit, LOC693049 ELISA Kit, LOC443015 ELISA Kit, Tubb5 ELISA Kit
    Background
    A Tubulin is one of several members of a small family of globular proteins. The most common members of the tubulin family are alpha-tubulin and beta-tubulin, the proteins that make up microtubules. Each has a molecular weight of approximately 55 kiloDaltons. Microtubules are assembled from dimers of alpha- and beta-tubulin. These subunits are slightly acidic with an isoelectric point between 5.2 and 5.8. Tubulin was long thought to be specific to eukaryotes. Recently, however, the prokaryotic cell division protein FtsZ was shown to be evolutionarily related to tubulin. To form microtubules, the dimers of alpha- and beta-tubulin bind to GTP and assemble onto the (+) ends of microtubules while in the GTP-bound state. After being incorporated into the microtubule, the bound molecule of GTP will hydrolyse into GDP. Although both subunits bind GTP, only the beta-subunit has GTPase activity, that is, beta-tubulin can hydrolyse GTP to GDP whereas alpha-tubulin cannot. Whether the beta-tubulin member of the tubulin dimer is bound to GTP or GDP influences the stability of the dimer in the microtubule. Dimers bound to GTP tend to assemble into microtubules, while dimers bound to GDP tend to fall apart, thus, this GTP cycle is essential for the dynamic instability of the microtubule. Class III beta-tubulin is a microtubule element expressed exclusively in neurons, and is a popular identifier specific for neurons in nervous tissue. Katanin is a protein complex that severs beta-tubulin, and is necessary for rapid microtubule transport in neurons and in higher plants.
    Pathways
    Microtubule Dynamics, M Phase
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