Preparation And Storage
Recommended Assay Procedures
Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Western blot: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/monoclonal_anti.jsp
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- Triton is a trademark of the Dow Chemical Company.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
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Tubulin is a highly conserved protein with a molecular weight of ~50 kD. The self-assembly of tubulin leads to microtubules, hollow cylinders that are one of the major components of the eukaryotic cytoskeleton. Microtubules play key roles in chromosome segregation in mitosis, intracellular transport, ciliary and flagellar bending, and structural support of the cytoskeleton. There are two main classes of tubulin isoforms, α- and β-tubulin, which are usually products of separate genes. Microtubules are made from protofilaments, strings of alternating α- and β-tubulin spaced 4 nm apart and pointing in the same direction. Tubulin can be posttranslationally modified in several ways, including phosphorylation, acetylation, glutamylation, and detyrosination. For example, microtubules that turn over slowly tend to be acetylated and detyrosinated.
The 5H1 monoclonal antibody reacts with β-tubulin. It does not cross-react with α-tubulin.
Development References (5)
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Brown KD, Wood KW, Cleveland DW. The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A. J Cell Sci. 1996; 109:961-969. (Biology).
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Cho J-H, Johnson GVW. Primed phosphorylation of tau at Thr231 by glycogen synthase kinase 3β (GSK3β) plays a critical role in regulating tau's ability to bind and stabilize microtubules. J Neurochem. 2004; 88:349-358. (Biology).
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Helfand BT, Mikami A, Vallee RB, Goldman RD. A requirement for cytoplasmic dynein and dynactin in intermediate filament network assembly and organization. J Cell Biol. 2002; 157(5):795-806. (Biology).
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Prahlad V, Yoon M, Moir RD, Vale RD, Goldman RD. Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks. J Cell Biol. 1998; 143(1):159-170. (Biology).
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Wang Y, Loomis PA, Zinkoswski RP, Binder LI. A novel tau transcript in cultured human neuroblastoma cells expressing nuclear tau. J Cell Biol. 1993; 121(2):257-267. (Biology).
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
