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Purified Mouse anti-Mouse Nanog
Purified Mouse anti-Mouse Nanog

Western blot analysis of Nanog expression on mouse embryonic stem cell lysate. Mouse lysate was probed with Purified Mouse anti-Mouse Nanog (Cat. No. 560259) at 2.0 (lane 1), 1.0 (lane 2), and 0.5 (lane 3) µg/ml, followed by HRP Goat Anti-Mouse Ig (Cat. No. 554002). Nanog is identified as a 42 kDa band.

Purified Mouse anti-Mouse Nanog

Immunofluorescent staining of mouse embryonic stem (ES) cell line.  ES-E14TG2a cells (ATCC CRL-1821) were seeded in a 96-well imaging plate at ~10,000 cells per well.  After overnight incubation, the cells were fixed, permeabilized with Triton™ X-100, and stained with Mouse anti-Mouse Nanog antibody according to the Recommended Assay Procedure.   The second step reagent was Alexa Fluor® 555 goat anti-mouse Ig (Invitrogen) (pseudo colored green). Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue).  The images were captured on a BD Pathway™ 435 High-Content Bioimager System 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.

Western blot analysis of Nanog expression on mouse embryonic stem cell lysate. Mouse lysate was probed with Purified Mouse anti-Mouse Nanog (Cat. No. 560259) at 2.0 (lane 1), 1.0 (lane 2), and 0.5 (lane 3) µg/ml, followed by HRP Goat Anti-Mouse Ig (Cat. No. 554002). Nanog is identified as a 42 kDa band.

Immunofluorescent staining of mouse embryonic stem (ES) cell line.  ES-E14TG2a cells (ATCC CRL-1821) were seeded in a 96-well imaging plate at ~10,000 cells per well.  After overnight incubation, the cells were fixed, permeabilized with Triton™ X-100, and stained with Mouse anti-Mouse Nanog antibody according to the Recommended Assay Procedure.   The second step reagent was Alexa Fluor® 555 goat anti-mouse Ig (Invitrogen) (pseudo colored green). Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue).  The images were captured on a BD Pathway™ 435 High-Content Bioimager System 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.

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BD Pharmingen™
Mouse (QC Testing)
Mouse BALB/c IgG1, κ
Mouse Nanog Recombinant Protein
Bioimaging, Western blot (Routinely Tested)
42 kDa
0.5 mg/ml
AB_1645261
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


推荐的实验流程

For more information on Western Blotting and Bioimaging applications, please refer to "Application resources" at our website: www.bdbiosciences.com/us/s/resources.

Recommended Protocol for Bioimaging:

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a 96-well Imaging Plate and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, and wash (1-2 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 (1-2 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 1-2 times with 100 μl of appropriate buffer (either 1× PBS or 1× Perm/Wash buffer, see step 5c.).

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.  

商品通知

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Triton is a trademark of the Dow Chemical Company.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
  8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560259 Rev. 2
抗体详情
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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.

560259 Rev. 2
格式详情
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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
560259 Rev.2
报价单和参考
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研发参考 (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). 查看参考
  2. Chambers I. The molecular basis of pluripotency in mouse embryonic stem cells. Cloning Stem Cells. 2004; 6(4):386-391. (Biology). 查看参考
  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). 查看参考
  4. Pan G, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 2007; 17:42-49. (Biology). 查看参考
  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). 查看参考
  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). 查看参考
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560259 Rev. 2

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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.