alpha Tubulin / TUBA1B (Tyr-Tubulin) antibody

Details for Product No. ABIN153391
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Antigen
Reactivity
Bird (Avian), Mammalian, Yeast
(1)
Host
Rat
(1)
Clonality (Clone)
Monoclonal ()
Application
Western Blotting (WB), ELISA, Immunocytochemistry (ICC), Immunofluorescence (IF), Immunohistochemistry (IHC), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP), Radioimmunoassay (RIA)
(1), (1), (1), (1)
Pubmed 5 references available
Quantity 0.1 mL
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Catalog No. ABIN153391
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Immunogen Full length native protein (purified) (S. cerevisiae).
Clone YL1-2
Isotype IgG2a
Cross-Reactivity (Details) Expected to react with most eukaryotes due to sequence identity. Drosophila reactivity reported in scientific literature (PMID: 24019759) Other species have not been tested.
Purification Protein G purified
Background This antibody can be used as a loading control on Western blots (Allen et al.) and is notdetected by anti-mouse Ig secondaries.It has been used in epitope tagging proceduresto detect proteins tagged with a C-terminal Gly-Gly-Phe(OH) epitope. Under somecircumstances this antibody may cross-react with other protein including E. coli rec Aand oxidized actin. Alternate Names: anti-Alpha tubulin ubiquitous antibody, anti-H2 ALPHA antibody, anti-TUBA1 antibody,anti-Tubulin alpha 1 chain antibody, anti-Tubulin alpha antibody, anti-Tubulin alpha ubiquitous chain antibody, anti-Tubulin K alpha 1 antibody.
Gene Symbol: TUBA1A
Gene ID 7846
Application Notes This Tubulin Antibody (YL1/2) is useful for Western blot, Immunoprecipitation, Immunohistochemistry on frozen and paraffin-embedded sections, Immunocytochemistry/Immunofluorescence, Radioimmunoassay and ELISA. NB600-506 is ideal for use as a Western blot loading control, where a band can be seen around 50-55 kDa and as a cytoskeletal marker in Immunocytochemistry.
Recommended dilutions: ELISA 1:100-1:1000, Immunocytochemistry/Immunofluorescence 1:1000-1:10000, Immunohistochemistry 1:200, Immunohistochemistry-Frozen 1:200, Immunohistochemistry-Paraffin 1:200, Immunoprecipitation 1:10-1:500, Radioimmunoassay, Western Blot 1:5000-1:10000
Protocol Protocol Specific for NB600-506 Western Blot Protocol
1. Perform SDS-PAGE on samples to be analyzed, loading 40 µg of total protein per lane.
. Transfer proteins to membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
. Rinse the blot.
. Block the membrane using standard blocking buffer for at least 1 hour.
. Wash the membrane in wash buffer three times for 10 minutes each.
. Dilute primary antibody in blocking buffer and incubate 1 hour at room temperature.
. Wash the membrane in wash buffer three times for 10 minutes each.
. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as required to reduce background).
. Apply the detection reagent of choice in accordance with the manufacturers instructions.Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2 %.Immunohistochemistry-Paraffin Embedded SectionsAntigen Unmasking:Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.
0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes. Staining:
. Wash sections in deionized water three times for 5 minutes each.
. Wash sections in wash buffer for 5 minutes.
. Block each section with 100-400 µL blocking solution for 1 hour at room temperature.
. Remove blocking solution and add 100-400 µL diluted primary antibody. Incubate overnight at 4 C.
. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
. Add 100-400 µL biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
. Add 100-400 µL Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
. Wash sections three times in wash buffer for 5 minutes each.
. Add 100-400 µL DAB substrate to each section and monitor staining closely.
. As soon as the sections develop, immerse slides in deionized water.
. Counterstain sections in hematoxylin.
. Wash sections in deionized water two times for 5 minutes each.
. Dehydrate sections.
. Mount coverslips.Immunocytochemistry ProtocolCulture cells to appropriate density in 35 mm culture dishes or 6-well plates.
. Remove culture medium and add 10 % formalin to the dish. Fix at room temperature for 30 minutes.
. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
. To block nonspecific antibody binding incubate in 10 % normal goat serum from 1 hour to overnight at room temperature.
. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room
Restrictions For Research Use only
Format Liquid
Concentration 1.0 mg/mL
Buffer Tris-glycine, 150 mM NaCl, Sodium Azide
Preservative Sodium azide
Precaution of Use WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.
Handling Advice Avoid freeze-thaw cycles
Storage 4 °C
Storage Comment 4 °C short term. Aliquot and store at -20 °C long term.
Supplier Images
anti-alpha Tubulin / TUBA1B (Tyr-Tubulin) antibody Western blot using ABIN153391. Lane 1 and 2: HeLa whole cell lysate, Lane 3 and 4: 3T3 (mouse) cell lysate.
General Breitling, Duebel, Seehaus et al.: "A surface expression vector for antibody screening." in: Gene, Vol. 104, Issue 2, pp. 147-53, 1991 (PubMed).

Clayton, Lloyd: "The relationship between the division plane and spindle geometry in Allium cells treated with CIPC and griseofulvin: an anti-tubulin study." in: European journal of cell biology, Vol. 34, Issue 2, pp. 248-53, 1984 (PubMed).

Kilmartin, Wright, Milstein: "Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line." in: The Journal of cell biology, Vol. 93, Issue 3, pp. 576-82, 1982 (PubMed).

Allen, Brinkhuis, van Deemter et al.: "Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance." in: Cancer research, Vol. 60, Issue 20, pp. 5761-6, 2000 (PubMed).

Iida, Hirota, Morisaki et al.: "Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G1 tetraploidy checkpoint function." in: Oncogene, Vol. 23, Issue 31, pp. 5266-74, 2004 (PubMed).

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