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Purified Mouse Anti-Metablastin
Purified Mouse Anti-Metablastin

Western blot analysis of Metablastin on a Jurkat lysate (Cat. No. 611451). Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the Metablastin (Stathmin) antibody.

Purified Mouse Anti-Metablastin

Immunofluorescent staining of HeLa cells (ATCC CCL-2).  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the Triton™ X-100 perm protocol and the anti- Metablastin antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective.  This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells and can be used with either perm protocol (see Recommended Assay Procedure).

Western blot analysis of Metablastin on a Jurkat lysate (Cat. No. 611451). Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the Metablastin (Stathmin) antibody.

Immunofluorescent staining of HeLa cells (ATCC CCL-2).  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the Triton™ X-100 perm protocol and the anti- Metablastin antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective.  This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells and can be used with either perm protocol (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Stathmin, Metablastin
Human (QC Testing), Rat (Tested in Development)
Mouse IgG2b
Human Metablastin aa. 38-147
Western blot (Routinely Tested), Bioimaging (Tested During Development)
19 kDa
250 µg/ml
AB_398686
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

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/pharmingen/protocols/Western_Blotting.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. 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.
  4. 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.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Triton is a trademark of the Dow Chemical Company.
611146 Rev. 4
Antibody Details
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53/Metablastin

The regulation of microtubule (MT) assembly is vital to cellular processes such as organelle transport, organization of the cytoplasm, and intracellular movement of cell surface receptors. MTs are composed of tubulin subunits that exist in dynamic equilibrium between free tubulin dimers and MTs. The instability of MTs is determined by the rates of growth and shrinkage of tubulin polymers and by frequencies of transitions from growth to shrinkage (catastrophes) or from shrinkage to growth (rescues). The most well known MT regulators are the microtubule associated proteins (MAPs) which directly bind and stabilize MTs. Metablastin (stathmin) opposes MAP activity by inducing catastrophes.Metablastin is variably phosphorylated on multiple Ser residues by kinases that are regulated by the cell cycle or by external signals. Phosphorylation of metablastin inhibits its ability to destabilize MTs and, in turn, induces tubulin polymerization. Metablastin activity is turned off during the cell cycle to allow spindle formation and cell division. Thus, metablastin is thought to function to regulate the dynamics of MT formation in response to external signals during the interphase of the cell cycle.

611146 Rev. 4
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
611146 Rev.4
Citations & References
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Development References (3)

  1. Gradin HM, Larsson N, Marklund U, Gullberg M. Regulation of microtubule dynamics by extracellular signals: cAMP-dependent protein kinase switches off the activity of oncoprotein 18 in intact cells. J Cell Biol. 1998; 140(1):131-141. (Biology). View Reference
  2. Horwitz SB, Shen HJ, He L, et al. The microtubule-destabilizing activity of metablastin (p19) is controlled by phosphorylation. J Biol Chem. 1997; 272(13):8129-8132. (Biology). View Reference
  3. Larsson N, Marklund U, Gradin HM, Brattsand G, Gullberg M. Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis. Mol Cell Biol. 1997; 17(9):5530-5539. (Biology). View Reference
611146 Rev. 4

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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.