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66871
4F2hc/CD98 (D3F9D) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

4F2hc/CD98 (D3F9D) XP® Rabbit mAb (PE Conjugate) #66871

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of SH-SY5Y cells (blue) and HeLa cells (green) using 4F2hc/CD98 (D3F9D) XP® Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry, Extracellular Epitope Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100mL 1X PBS. Store at 4°C.

B. Fixation

NOTE: If live cell staining is desired, proceed to Section C.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml PBS. Add formaldehyde to obtain a final concentration of 4% formaldehyde.
  3. Fix for 15 minutes at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.
  5. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells.
  2. If necessary, centrifuge to remove excess PBS.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 30-60 minutes at room temperature.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to Section D.

D. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted January 2009

revised June 2017

Protocol Id: 180

Application Dilutions
Flow Cytometry 1:50
Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

4F2hc/CD98 (D3F9D) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total 4F2hc/CD98 protein. This antibody is predicted to detect multiple isoforms of 4F2hc/CD98.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val509 of human 4F2hc/CD98 protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated 4F2hc/CD98 (D3F9D) XP® Rabbit mAb #47213.

4F2hc is a transmembrane protein that belongs to the solute carrier family. 4F2hc forms heterodimeric complexes with various amino acid transporters such as LAT1 and LAT2 and regulates uptake of amino acids (1-5). 4F2hc is one of the earliest expressed antigens on the surface of activated human lymphocytes (6), hence it is also named CD98. 4F2hc is expressed in all cell types with the exception of platelets, and is expressed at highest levels in the tubules of the kidney and the gastrointestinal tract (7,8). It is localized at the plasma membrane when associated with LAT1 or LAT2 (9) and at the apical membrane of placenta (10). Research studies have shown that 4F2hc is highly expressed in various tumors including glioma (11), ovarian cancer (12), and astrocytomas (13), and it has been implicated in tumor progression and correlated with poor outcome in patients with pulmonary neuroendocrine tumors (14). 4F2hc is also involved in integrin trafficking through association with β1 and β4 integrins, and regulates keratinocyte adhesion and differentiation (15).

  1. Kanai, Y. et al. (1998) J Biol Chem 273, 23629-32.
  2. Mastroberardino, L. et al. (1998) Nature 395, 288-91.
  3. Pfeiffer, R. et al. (1999) EMBO J 18, 49-57.
  4. Pineda, M. et al. (1999) J Biol Chem 274, 19738-44.
  5. Sato, H. et al. (1999) J Biol Chem 274, 11455-8.
  6. Lumadue, J.A. et al. (1987) Proc Natl Acad Sci USA 84, 9204-8.
  7. Rossier, G. et al. (1999) J Biol Chem 274, 34948-54.
  8. Dave, M.H. et al. (2004) J Physiol 558, 597-610.
  9. Bröer, A. et al. (2001) Biochem J 355, 725-31.
  10. Ritchie, J.W. and Taylor, P.M. (2001) Biochem J 356, 719-25.
  11. Okubo, S. et al. (2010) J Neurooncol 99, 217-25.
  12. Kaji, M. et al. (2010) Int J Gynecol Cancer 20, 329-36.
  13. Nawashiro, H. et al. (2006) Int J Cancer 119, 484-92.
  14. Kaira, K. et al. (2011) Oncol Rep 26, 931-7.
  15. Lemaître, G. et al. (2011) J Dermatol Sci 61, 169-79.
Entrez-Gene Id
6520
Swiss-Prot Acc.
P08195
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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製品番号 サイズ 価格 在庫
66871S
100 µl  (50 tests)

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