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Alexa Fluor® 647 Mouse anti-β-Tubulin 5H1 RUO

Alexa Fluor® 647 Mouse anti-β-Tubulin 

Clone  5H1   (RUO)

  • Brand BD Pharmingen™
  • Vol. Per Test 5 µl
  • Isotype Mouse IgM, κ
  • Reactivity Human, Rat (QC Testing)  Mouse, Syrian Hamster, Chinese Hamster, Indian Muntjac (Reported) 
  • Application Bioimaging (Routinely Tested) 
  • Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
  • Regulatory Status RUO
Product Details Recommended Assay References

Description

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.

Format
  • Format Alexa Fluor® 647
  • Excitation Source Red 633 nm
  • Excitation Max 650 nm
  • Emission Max 668 nm

Alexa Fluor® 647 conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.

Other Formats →

Suggested Companion Products

Stain Buffer (FBS) RUO
500 mL Cat No: 554656

Perm Buffer III RUO
125 mL Cat No: 558050

Fixation Buffer RUO
100 mL Cat No: 554655

Resources & Tools
 Spectrum Viewer  Panel Designer  Spectrum Viewer  Download TDS  Regulatory Document Website
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.

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/pharmingen/protocols for technical protocols.


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.


  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.

  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.

  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.

  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.

  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.

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