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68591
Lamin B1 (119D5-F1) Mouse mAb

Lamin B1 (119D5-F1) Mouse mAb #68591

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Mouse IgG1
IF-IC

Confocal immunofluorescent analysis of HT-29 cells using Lamin B1 (119D5-F1) Mouse mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 647 Conjugate) #8584 (red). Blue pseudocolor = Propidium Iodide (PI)/RNase Staining Solution #4087 (fluorescent DNA dye).

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Methanol, 100%
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with ice-cold 100% methanol.
  2. Allow cells to fix for 15 minutes at -20°C.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  7. Rinse in 1X PBS as in step 5.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  9. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted December 2010

Protocol Id: 145

Aliquot into single-use vials to avoid multiple freeze/thaw cycles and store at -20°C. A slight precipitate may be present, but will not interfere with antibody performance.

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:200
Storage:

Supplied at 1 mg/mL in PBS containing 0.09% sodium azide. Store at -20°C. The product will freeze at -20°C.

Lamin B1 (119D5-F1) Mouse mAb recognizes endogenous levels of total Lamin B1 by immunofluroescence. Testing by immunofluorescence was validated for human cells. Reactivity is expected in other species, including mouse and rat.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with purified rat liver lamins.

Lamins are nuclear membrane structural components that are important in maintaining normal cell functions, such as cell cycle control, DNA replication, and chromatin organization (1-3). Lamins have been subdivided into types A and B. Type-A lamins consist of lamin A and C, which arise from alternative splicing of the lamin A gene LMNA. Lamin A and C are cleaved by caspases into large (41-50 kDa) and small (28 kDa) fragments, which can be used as markers for apoptosis (4,5). Type-B lamins consist of lamin B1 and B2, encoded by separate genes (6-8). Lamin B1 is also cleaved by caspases during apoptosis (9). Research studies have shown that duplication of the lamin B1 gene LMNB1 is correlated with pathogenesis of the neurological disorder adult-onset leukodystrophy (10).

  1. Gruenbaum, Y. et al. (2000) J Struct Biol 129, 313-23.
  2. Goldberg, M. et al. (1999) Crit Rev Eukaryot Gene Expr 9, 285-93.
  3. Yabuki, M. et al. (1999) Physiol Chem Phys Med NMR 31, 77-84.
  4. Rao, L. et al. (1996) J Cell Biol 135, 1441-55.
  5. Orth, K. et al. (1996) J Biol Chem 271, 16443-6.
  6. Biamonti, G. et al. (1992) Mol Cell Biol 12, 3499-506.
  7. Lin, F. and Worman, H.J. (1995) Genomics 27, 230-6.
  8. Pollard, K.M. et al. (1990) Mol Cell Biol 10, 2164-75.
  9. Chandler, J.M. et al. (1997) Biochem J 322 ( Pt 1), 19-23.
  10. Padiath, Q.S. et al. (2006) Nat Genet 38, 1114-23.
Entrez-Gene Id
4001
Swiss-Prot Acc.
P20700
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation, for research use only for immunocytometry, immunohistochemistry, high content screening (HCS) analysis, or flow cytometry applications.
The transfer of this product is contingent on the buyer using the purchased product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity), for Immunocytochemistry, high content screening (HCS) analysis, or flow cytometry applications. The sale of this product is expressly conditioned on the buyer not using the product or its components (1) in manufacturing; (2) to provide a service, information, or data to an unaffiliated third party for payment; (3) for therapeutic, diagnostic or prophylactic purposes; (4) resale, whether or not such product or its components are resold for use in research; or for any other commercial purpose. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

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Product # Size Price
68591S
100 µg $ 255.0

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