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Purified Mouse Anti-Ki-67
Product Details
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BD Pharmingen™
MKI67; Antigen identified by monoclonal antibody Ki-67; KIA
Human (QC Testing), Mouse (Tested in Development), Rat, Rhesus (Reported)
Mouse IgG1, κ
Human Ki-67
Bioimaging, Intracellular staining (flow cytometry) (Routinely Tested), Western blot (Not Recommended)
0.5 mg/ml
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.

Recommended Assay Procedures

STAINING PROTOCOL FOR FLOW CYTOMETRY:

1.  Harvest, count and pellet cells following standard procedures (Note: Ki-67 is expressed by the proliferative cells. You may get no staining with the resting cells, e.g., unstimulated PBMC).

2.  While votexing, add 5 ml drop by drop of cold 70-80% ethanol into the cells pellet (1-5x10^7 cells). Then incubate at -20°C for 2 hours minimum. These fixed cells can be used up to 60 days after fixing (store at -20°C).

3.  Add 30-40 ml wash buffer (PBS with 1% FBS, 0.09% NaN3, pH 7.2) to the fixed cells. Centrifuge the cells for 10 minutes at 1000 rpm and aspirate supernatant. Wash one more time with 30-40 ml wash buffer. Centrifuge at 1000 rpm for 10 minutes and aspirate supernatant.

4.  Resuspend the cells to a concentration of 1 x 10^7/ml (1 x 10^6/100 µl).

5.  Transfer 100 µl cell suspension into each fresh tube.

6.  Add 20 µl of properly diluted antibody according to the protocol into the tubes above. Mix gently.

7.  Incubate the tubes at room temperature (RT) for 20-30 minutes in the dark.

8.  Wash with 2 ml of PBS washing buffer at 1000 rpm for 5 minutes.

9.  Aspirate the supernatant.

10. For direct conjugated antibody: go to steps 13 & 14.

11. For purified antibody: add 50 µl of diluted secondary antibody at optimal concentration (Cat. No. 555988), incubate at RT for 30 minutes in the dark.

12. Repeat step 8 & 9.

13. Add 0.5 ml of PBS wash buffer into each tube. For FITC conjugated antibody, add µl of PI Staining Solution (Cat. No. 556463); for PE-conjugated antibody, add 20 µl BD Via-Probe™ Cell Viability Solution (Cat. No. 555816) into each tube.

14. Analyze the sample with FACS.

STAINING PROTOCOL FOR BIOIMAGING:

Methanol Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and

add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking

buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at

RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image

sample.

Triton-X 100 Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and

add 100 µl/well 0.1% Triton™ X-100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well

blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1

hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three

times with PBS. Image sample.

Product Notices

  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. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  5. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  6. 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.
  7. Triton is a trademark of the Dow Chemical Company.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
556003 Rev. 9
Antibody Details
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B56

The B56 monoclonal antibody specifically binds to the Ki-67 antigen that is expressed in the nucleus of cycling cells (G1, S, G2, M cell cycle phases). During the G0 phase, the antigen cannot be detected. During interphase of the cell cycle, it is associated with nucleolar components, and it is on the surface of the chromosomes during M phase. Ki-67 is a large protein having 2 alternatively spliced isoforms, an N-terminal forkhead-associated domain, a C-terminal domain that binds to heterochromatin proteins, and multiple phosphorylation sites, the functions of which are still unclear. Because of the strict association of Ki-67 expression with cell proliferation, anti-Ki-67 antibodies are useful for the identification, quantification, and monitoring of growing cell populations.

556003 Rev. 9
Format Details
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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
556003 Rev.9
Citations & References
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Development References (14)

  1. Benson MJ, Elgueta R, Schpero W, et al. Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals. J Exp Med. 2009; 206(9):2013-2025. (Clone-specific: Flow cytometry). View Reference
  2. Bigley V, Haniffa M, Doulatov S, et al. The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency. J Exp Med. 2011; 208(2):227-234. (Clone-specific: Flow cytometry). View Reference
  3. Bruno S, Crissman HA, Bauer KD, Darzynkiewicz Z. Changes in cell nuclei during S phase: progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34. Exp Cell Res. 1991; 196(1):99-106. (Biology: Flow cytometry). View Reference
  4. Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif. 1992; 25(1):31-40. (Biology: Flow cytometry). View Reference
  5. Kill IR. Localisation of the Ki-67 antigen within the nucleolus: evidence for a fibrillarin-deficient region of the dense fibrillar component. J Cell Sci. 1996; 109(6):1253-1263. (Biology). View Reference
  6. Kouro T, Medina KL, Oritani K, Kincade PW. Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators. Blood. 2001; 97(9):2708-2715. (Clone-specific: Flow cytometry). View Reference
  7. Kubbutat MH, Key G, Duchrow M, Schluter C, Flad HD, Gerdes J. Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein). J Clin Pathol. 1994; 47(6):524-528. (Biology). View Reference
  8. Picker LJ, Hagen SI, Lum R, et al. Insufficient production and tissue delivery of CD4+ memory T cells in rapidly progressive simian immunodeficiency virus infection. J Exp Med. 2004; 200(10):1299-1314. (Clone-specific: Flow cytometry). View Reference
  9. Pitcher CJ, Hagen SI, Walker JM, et al. Development and homeostasis of T cell memory in rhesus macaque. J Immunol. 2002; 168(1):29-43. (Clone-specific: Flow cytometry). View Reference
  10. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown.. J Cell Physiol. 2000; 182(3):311-22. (Biology). View Reference
  11. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991; 39(6):741-748. (Biology). View Reference
  12. Spargo LDJ, Cleland LG, Cockshell MP, Mayrhofer Graham. Recruitment and proliferation of CD4+ T cells in synovium following adoptive transfer of adjuvant-induced arthritis. Int Immunol. 2006; 18(6):897-910. (Clone-specific: Flow cytometry, Immunofluorescence).
  13. Starborg M, Gell K, Brundell E, Höög C. The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression. J Cell Sci. 1996; 109(1):143-153. (Biology). View Reference
  14. Valenti LM, Mathieu J, Chancerelle Y, et al. High levels of endogenous nitric oxide produced after burn injury in rats arrest activated T lymphocytes in the first G1 phase of the cell cycle and then induce their apoptosis. Exp Cell Res. 2005; 306(1):150-167. (Clone-specific: Flow cytometry). View Reference
View All (14) View Less
556003 Rev. 9

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