LEFT: Western blot analysis of Nkx2.2 expression in human embryonic kidney cell lysate. Lysates of 293-F cells (Life Technologies R790-07) were prepared for electrophoresis (SDS-PAGE) in a 2D Tris-Glycine polyacrylamide gel. The proteins were transferred to PVDF membranes and then probed with 0.5, 0.25, 0.125, 0.062 µg/mL (Lanes 1-4, respectively) of Purified Mouse Anti-Nkx2.2. Specific staining was detected with HRP Goat Anti-Mouse Ig (Cat. No. 554002). RIGHT: Flow cytometric analysis of Nkx2.2 expression in human embryonic kidney cells. 293-F cells (Life Technologies R790-07) were fixed and permeabilized with the BD Pharmingen™ Transcription Factor Buffer Set (Cat No. 562574). The fixed cells were then stained with either Purified Mouse IgG2b, κ Isotype Control (Cat. No. 556654, dashed line) or Purified Mouse Anti-Nkx2.2 (solid line). Specific staining was detected with PE Goat Anti-Mouse Ig (Cat. No. 550589). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry was performed using a BD FACSCanto™ II Flow Cytometer System.
LEFT: Immunohistochemical staining Nkx2.2 in human pancreas. Following antigen retrieval with BD Retrievagen A Buffer (Cat. No. 550524), the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b κ Isotype Control (Cat. No.556654, left) or Purified Mouse Anti-Nkx2.2 (right). A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin (Cat. No. 550337), Streptavidin HRP (Cat. No.550946), and the DAB Substrate Kit (Cat. No. 550880) was used to develop the primary staining reagents. Original magnification: 40×. RIGHT: Immunofluorescent staining of Nkx2.2 in mouse insulinoma cells. Beta-TC-6 cells (ATCC CRL-11506) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and stained with Purified Mouse Anti-Nkx2.2 at 1.2 μg/mL. The second-step reagent was Alexa Fluor® 488 Goat Anti-Mouse Ig (Life Technologies, pseudo-colored green), and the counter-staining was with DAPI (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ software. Permeabilization with BD Perm/Wash™ Buffer (Cat No. 554723) or 0.1% Triton™ X-100 buffer is also suitable for use with this antibody.
LEFT: Western blot analysis of Nkx2.2 expression in human embryonic kidney cell lysate. Lysates of 293-F cells (Life Technologies R790-07) were prepared for electrophoresis (SDS-PAGE) in a 2D Tris-Glycine polyacrylamide gel. The proteins were transferred to PVDF membranes and then probed with 0.5, 0.25, 0.125, 0.062 µg/mL (Lanes 1-4, respectively) of Purified Mouse Anti-Nkx2.2. Specific staining was detected with HRP Goat Anti-Mouse Ig (Cat. No. 554002). RIGHT: Flow cytometric analysis of Nkx2.2 expression in human embryonic kidney cells. 293-F cells (Life Technologies R790-07) were fixed and permeabilized with the BD Pharmingen™ Transcription Factor Buffer Set (Cat No. 562574). The fixed cells were then stained with either Purified Mouse IgG2b, κ Isotype Control (Cat. No. 556654, dashed line) or Purified Mouse Anti-Nkx2.2 (solid line). Specific staining was detected with PE Goat Anti-Mouse Ig (Cat. No. 550589). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry was performed using a BD FACSCanto™ II Flow Cytometer System.
LEFT: Immunohistochemical staining Nkx2.2 in human pancreas. Following antigen retrieval with BD Retrievagen A Buffer (Cat. No. 550524), the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b κ Isotype Control (Cat. No.556654, left) or Purified Mouse Anti-Nkx2.2 (right). A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin (Cat. No. 550337), Streptavidin HRP (Cat. No.550946), and the DAB Substrate Kit (Cat. No. 550880) was used to develop the primary staining reagents. Original magnification: 40×. RIGHT: Immunofluorescent staining of Nkx2.2 in mouse insulinoma cells. Beta-TC-6 cells (ATCC CRL-11506) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), and stained with Purified Mouse Anti-Nkx2.2 at 1.2 μg/mL. The second-step reagent was Alexa Fluor® 488 Goat Anti-Mouse Ig (Life Technologies, pseudo-colored green), and the counter-staining was with DAPI (pseudo-colored blue). The images were captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ software. Permeabilization with BD Perm/Wash™ Buffer (Cat No. 554723) or 0.1% Triton™ X-100 buffer is also suitable for use with this antibody.
Product Details
BD Pharmingen™
NKX2-2; NKX2B; Nkx2-2; Nkx-2.2; NK-2 homolog B; glycoprotein GP330
Human (QC Testing), Mouse (Tested in Development), Rat,Chicken (Reported)
Mouse BALB/c IgG2b, κ
Chicken Nkx2.2 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested), Bioimaging, Immunofluorescence, Immunohistochemistry-formalin (antigen retrieval required), Western blot (Tested During Development)
30-40 kDa
0.5 mg/ml
AB_2738924
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- 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.
- 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.
- Triton is a trademark of the Dow Chemical Company.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Transcription Factor Buffer Set RUO
Size
100 Tests
Cat No.
562574
564731 Rev. 1
Antibody Details
74.5A5
The 74.5A5 monoclonal antibody specifically binds to Homeobox protein Nkx-2.2 (Nkx2.2), which is a transcription factor required for the differentiation of ventral neuronal populations in the central nervous system and endocrine cell populations in the pancreas and intestine. Within the pancreas, Nkx2.2 is expressed in many cell subsets and plays important roles in islet and β-cell differentiation and maturation. Mice with a homozygous disruption of the Nkx2-2 gene lack mature β cells and die shortly after birth with severe hyperglycemia. Similarly to the Nkx2.2-ablated mice, homozygous mutations in the human NKX2-2 gene have been described in diabetic patients and associated with the etiology of neonatal diabetes. The NKX2-2 gene is also a target of the Ewing sarcoma specific protein EWS-FLI. Nkx2.2 expression can be monitored in order to determine cell fate changes in cultures for the generation of human pluripotent stem cell-derived insulin-producing cells as well as oligodendrocyte progenitors.
564731 Rev. 1
Format Details
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
564731 Rev.1
Citations & References
Development References (6)
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Briscoe J, Sussel L, Serup P, et al. Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling.. Nature. 1999; 398(6728):622-7. (Clone-specific: Immunohistochemistry). View Reference
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D'Amour KA, Bang AG, Eliazer S, et al . Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. 2006; 24(12):1481-1483. (Clone-specific: Immunofluorescence, Western blot). View Reference
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Ericson J, Rashbass P, Schedl A, et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signalling. Cell. 1997; 90(1):169-180. (Immunogen: Immunohistochemistry). View Reference
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Sussel L, Kalamaras J, Hartigan-O'Connor DJ, et al. Mice Lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. Development. 1998; 125(12):2213-2221. (Clone-specific: Immunohistochemistry). View Reference
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Wang S, Bates J, Li X, et al. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell. 2013; 12(2):252-264. (Clone-specific: Immunofluorescence). View Reference
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Yoshida A, Sekine S, Tsuta K, Fukayama M, Furuta K, Tsuda H. NKX2.2 is a useful immunohistochemical marker for Ewing sarcoma. Am J Surg Pathol. 2012; 36(7):993-999. (Biology). View Reference
564731 Rev. 1
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.
