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Alexa Fluor® 647 Mouse anti-β-Tubulin
Alexa Fluor® 647 Mouse anti-β-Tubulin

Immunofluorescence staining of human cell lines.  U-2 OS cells (ATCC HTB-96) were cultured, fixed, permeabilized with cold methanol, stained with Alexa Fluor® 647 Mouse anti-β-Tubulin (pseudo-colored green) and counter-stained with Hoechst 33342 (pseudo-colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 855 Bioimager System with a 20x objective and merged using BD Attovision™ software.  This antibody also stains A549 (ATCC CCL-185) and HeLa (ATCC CCL-2) cells, and it works with either cold methanol or Triton X-100 permeabilization (see Recommended Assay Procedure).

Immunofluorescence staining of human cell lines.  U-2 OS cells (ATCC HTB-96) were cultured, fixed, permeabilized with cold methanol, stained with Alexa Fluor® 647 Mouse anti-β-Tubulin (pseudo-colored green) and counter-stained with Hoechst 33342 (pseudo-colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 855 Bioimager System with a 20x objective and merged using BD Attovision™ software.  This antibody also stains A549 (ATCC CCL-185) and HeLa (ATCC CCL-2) cells, and it works with either cold methanol or Triton X-100 permeabilization (see Recommended Assay Procedure).

Product Details
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BD Pharmingen™
Human, Rat (QC Testing), Mouse, Syrian Hamster, Chinese Hamster, Indian Muntjac (Reported)
Mouse IgM, κ
Bioimaging (Routinely Tested)
5 µl
AB_647147
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

Recommended Assay Procedures

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 fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).

3.        Remove the fixative from the wells, and permeabilize the cells using either cold methanol or Triton™ X-100:

a.        Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow 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.

Triton is a trademark of The Dow Chemical Company.

4.        Remove the permeabilizer, and wash the wells twice with 100 μl of 1× PBS.

5.        Remove the PBS, and block the cells by adding 100 µl of blocking buffer (3% FBS in 1× PBS) or BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well and incubating for 30 minutes at RT.

6.        Remove the blocking buffer, dilute the antibody conjugate 1:10 in blocking buffer or Stain Buffer (FBS), and stain the cells by adding 50 µl of the diluted antibody conjugate to each well and incubating for 1 hour at RT.

7.        Remove the diluted antibody conjugate, and wash the wells three times with 100 μl of 1× PBS.

8.        Remove the PBS, and counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

9.        View and analyze the cells on an appropriate imaging instrument.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
  2. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  3. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. 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.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558606 Rev. 2
Antibody Details
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5H1

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.

558606 Rev. 2
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
558606 Rev.2
Citations & References
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Development References (5)

  1. 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: Immunofluorescence).
  2. 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: Immunofluorescence).
  3. 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: Immunofluorescence).
  4. 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: Immunofluorescence).
  5. 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: Immunofluorescence).
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558606 Rev. 2

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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.