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Alexa Fluor® 488 Mouse anti-Mouse Nanog
Alexa Fluor® 488 Mouse anti-Mouse Nanog

Immunofluorescent staining of mouse embryonic stem cell line.  ES-E14TG2a cells (ATCC CRL-1821) were were seeded in a 96-well imaging plate (Cat. No. 353219) at ~10,000 cells per well.  After overnight incubation, the cells were fixed, permeabilized with Triton™ X-100, and stained with Alexa Fluor® 488 Mouse anti-Mouse Nanog (pseudo colored green) according to the Recommended Assay Procedure.  Cell nuclei were counter stained with Hoechst 33342 (pseudo colored blue).  The images were captured on a BD Pathway™ 435 Cell Analyzer using a 20X objective and merged using BD AttoVision™ software.  This antibody also stains F9 cells (mouse embryonal carcinoma, ATCC CRL-1720).  It also worked with the Saponin and cold methanol fix/perm protocols (see Recommended Assay Procedure), however Saponin permeabilization resulted in higher background staining.

Immunofluorescent staining of mouse embryonic stem cell line.  ES-E14TG2a cells (ATCC CRL-1821) were were seeded in a 96-well imaging plate (Cat. No. 353219) at ~10,000 cells per well.  After overnight incubation, the cells were fixed, permeabilized with Triton™ X-100, and stained with Alexa Fluor® 488 Mouse anti-Mouse Nanog (pseudo colored green) according to the Recommended Assay Procedure.  Cell nuclei were counter stained with Hoechst 33342 (pseudo colored blue).  The images were captured on a BD Pathway™ 435 Cell Analyzer using a 20X objective and merged using BD AttoVision™ software.  This antibody also stains F9 cells (mouse embryonal carcinoma, ATCC CRL-1720).  It also worked with the Saponin and cold methanol fix/perm protocols (see Recommended Assay Procedure), however Saponin permeabilization resulted in higher background staining.

Product Details
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BD Pharmingen™
Mouse (QC Testing)
Mouse BALB/c IgG1, κ
Mouse Nanog Recombinant Protein
Bioimaging (Routinely Tested)
5 µl
AB_1645191
Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, and wash (one to two times) with 100 μl of 1× PBS.

3.        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).

4.        Remove the fixative from the wells, and wash the wells (one to two times) with 100 μl of 1× PBS.

5.        Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c):

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.

b.        Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

c.        Add 100 µl of 1× Perm/Wash buffer (Cat. No. 554723) to each well and incubate for 15 to 30 minutes at RT.  Continue to use 1× Perm/Wash buffer for all subsequent wash and dilutions steps.

6.        Remove the permeabilization buffer from the wells, and wash one to two times with 100 μl of appropriate buffer (either 1× PBS or 1× Perm/Wash buffer, see step 5.c.).

7.        Optional blocking step:  Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.

8.        Dilute the antibody to its optimal working concentration in appropriate dilution buffer.  Titrate purified (unconjugated) antibodies and second-step reagents to determine the optimal concentration.  If using a Bioimaging Certified antibody conjugate, dilute it 1:10.

9.        Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT.  Incubate in the dark if using fluorescently labeled antibodies.

10.        Remove the antibody, and wash the wells three times with 100 μl of wash buffer.  An optional detergent wash (100 μl of 0.05% Tween in 1× PBS) can be included prior to the regular wash steps.

11.   If the antibody being used is fluorescently labeled, then move to step 12.  Otherwise, if using a purified unlabeled antibody, repeat steps 8 to 10 with a fluorescently labeled second-step reagent to detect the purified antibody.

12.        After the final wash, 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.

13.        View and analyze the cells on an appropriate imaging instrument.  Recommended filters for the BD Pathway™ instruments are:

Instrument                        Excitation        Emission        Dichroic

BD Pathway 855                488/10                515 LP                Fura/FITC

BD Pathway 435                482/35                536/40                FF506

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  3. 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.
  4. Triton is a trademark of the Dow Chemical Company.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
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Antibody Details
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M55-312

The M55-312 monoclonal antibody reacts with mouse Nanog (named for Tir Na Nog, the land of the ever-young of Celtic mythology), which is a homeobox transcription factor required for the maintenance of the undifferentiated state of pluripotent stem cells.  Nanog expression counteracts the differentiation-promoting signals induced by the extrinsic factors LIF (Leukemia Inhibitory Factor) and BMP (Bone Morphogenic Protein).  When Nanog expression is down-regulated, cell differentiation can proceed.  Proteins that regulate Nanog expression include transcription factors Oct4, SOX2, FoxD3, and Tcf3 and tumor suppressor p53.

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Format Details
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
560261 Rev.1
Citations & References
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Development References (6)

  1. Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003; 113:643-655. (Biology). View Reference
  2. Chambers I. The molecular basis of pluripotency in mouse embryonic stem cells. Cloning Stem Cells. 2004; 6(4):386-391. (Biology). View Reference
  3. Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 2003; 113:631-642. (Biology). View Reference
  4. Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 2007; 17:42-49. (Biology). View Reference
  5. Sun Y, Li H, Yang H, Rao MS, Zhan M. Mechanisms controlling embryonic stem cell self-renewal and differentiation. Crit Rev Eukaryot Gene Expr.. 2006; 16(3):211-231. (Biology). View Reference
  6. Suzuki A, Raya A, Kawakami Y, et al. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells. Proc Natl Acad Sci U S A. 2006; 103(27):10294-10299. (Biology). View Reference
View All (6) View Less
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Please refer to Support Documents for Quality Certificates


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.