For Research Use Only.
Not for use in diagnostic procedures.

566
Page 1 of 3

POLYCLONAL ANTIBODY

Anti-CD117 (c-Kit) (Human) pAb
Code No.
566

Quantity100 L

BACKGROUND: c-Kit, also known as stem cell factor
receptor, steel factor receptor or CD117 is classified as a
type III receptor tyrosine kinase (RTK) belonging to theplatelet-derived growth factor receptor subfamily. Bindingof stem cell factor (SCF), known as c-Kit ligand to c-Kit,
initiates autophosphorylation of the receptor, subsequently
leading to promotes a signal transduction cascade throughRas-Raf-MAP kinase cascade, phosphatidylinositol3-kinase, src family kinases, and JAK/STAT pathways. Theroles of c-Kit include maturation of hematopoietic andprimordial germ cells precursors and melanocytes during
embryonic development. In acute myeloid leukemia (AML),
c-Kit has been proposed to play a functional role, andbecomes target molecule for drug development.

SOURCE: This antibody was purified from rabbit serum
using the synthesized peptides (C-terminal of c-Kit geneproduct; K963) coupled protein A agarose column. Therabbit was immunized with carrier protein conjugatedsynthesized peptides (K963).

FORMULATION: 100 L volume of PBS containing

Form
Purified IgG

SPECIES CROSS REACTIVITY:
Species

HumanMouse

RatSamples

HEL,
pancreas

Not tested

Not tested

Reactivity on
IHC/IC+INTENDED USE:
For Research Use Only. Not for use in diagnostic procedures.

REFERENCES:
1) Enomoto, T., et al., Clin. J. Gasteroenterol. 3, 73-77 (2010) [IHC]2) Koch, A. C., et al., Ann. N.Y. Acad. Sci. 1073, 517-526 (2006) [IHC]
3) Wilczynski, S. P., et al., Hum. Pathol. 36, 242-249 (2005) [IHC]
4) Lyford, G. L., et al., Gut 51, 496-501 (2002) [IHC]
5) Sakurai, S., et al., Am. J. Pathol. 154, 23-28 (1999) [IHC]
6) Tsuura, T., et al., Virchows Archiv. 424, 135-141 (1994)
7) Hidi, K., et al., Oncogene 6, 2291-2296 (1991)
8) Yarden,Y., et al., EMBO J. 6, 3341-3351 (1987)

kDa

50% glycerol, pH 7.2. No preservative is contained.

200 -STORAGE: This antibody solution is stable for one year

116 97 -from the date of purchase when stored at -20°C.

REACTIVITY: This antibody reacts with c-Kit (14566-kDa) on Western blotting and Immunohistochemistry.

APPLICATIONS:
Western blotting; 1:1,000 for chemiluminescence detection
systemImmunoprecipitation; Not tested
Immunohistochemistry; 1:200
Heat treatment is necessary for paraffin embeddedsections.
Microwave oven; 2 times for 10 minutes each in 10 mM
citrate buffer (pH 6.5)
Immunocytochemistry; 1:200
Heat treatment is necessary for paraffin embeddedsections.
Microwave oven; 2 times for 10 minutes each in 10 mM
citrate buffer (pH 6.5)
Flow cytometry; Not tested
Detailed

procedure

PROTOCOLS.

isprovided

inthefollowing

45Western blot analysis of c-kit
expression in HEL cells using
566.

PROTOCOLS:SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10
volume of cold Lysis buffer [50 mM Tris-HCl (pH 7.2),
250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol]containing appropriate protease inhibitors. Incubate it at
4°C with rotating for 30 minutes, then sonicate briefly (upto 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°Cand transfer the supernatant to another tube. Measure theprotein concentration of the supernatant and add the cold

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL http://ruo.mbl.co.jp
e-mail support@mbl.co.jp, TEL 052-238-1904

566
Page 2 of 3

Lysis buffer to make 8 mg/mL solution.
3) Mix the sample with equal volume of Laemmli’s samplebuffer.
4) Boil the samples for 3 minutes and centrifuge. Load 10
L of the sample per lane in a 1 mm thick
SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF)
membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer
system (Transfer Buffer: 25 mM Tris, 190 mM glycine,
20% MeOH). See the manufacture's manual for precisetransfer procedure.
6) To reduce nonspecific binding, soak the membrane in
10% skimmed milk (in PBS, pH 7.2) for 1 hour at room
temperature, or overnight at 4°C.
7) Incubate the membrane with primary antibody diluted
with PBS, pH 7.2 containing 1% skimmed milk assuggest in the APPLICATIONS for 1 hour at room
temperature. (The concentration of antibody will dependon condition.)
8) Wash the membrane with PBS-T [0.05% Tween-20 inPBS] (5 minutes x 3 times).
9) Incubate the membrane with 1:10,000 of Anti-IgG(Rabbit) pAb-HRP (MBL; code no. 458) diluted with 1%
skimmed milk (in PBS, pH 7.2) for 1 hour at room
temperature.
10) Wash the membrane with PBS-T (10 minutes x 3 times).
11) Wipe excess buffer on the membrane, then incubate itwith appropriate chemiluminescence reagent for 1 minute.
12) Remove extra reagent from the membrane by dabbingwith paper towel, and seal it in plastic wrap.
13) Expose to an X-ray film in a dark room for 3 minutes.
Develop the film as usual. The condition for exposure anddevelopment may vary.
(Positive control for Western blotting; HEL)

Immunocytochemical detection ofc-Kit on paraffin embedded sectionof HEL cells with 566.
Immunocytochemical staining for paraffin-embedded
sections
1) Deparaffinize the sections with Xylene 3 times for 3-5
minutes each.
2) Wash the slides with Ethanol 3 times for 3-5 minuteseach.
3) Wash the slides with PBS 3 times for 3-5 minutes each.
4) Heat treatment

Heat treatment by microwave oven:
Place the slides put on staining basket in 500 mL beakerwith 500 mL citrate buffer (pH 6.5). Cover the beakerwith plastic wrap, then process the slides 2 times for 10minutes each at 500 W with microwave oven. Let theslides cool down in the beaker at room temperature forabout 40 minutes.
5) Remove the slides from the citrate buffer and cover eachsection with 3% H2O2 for 10 minutes at room temperature
to block endogenous peroxidase activity. Wash 3 times inPBS for 5 minutes each.
6) Remove the slides from PBS, wipe gently around eachsection and cover tissues with 1% BSA in PBS for 5
minutes to block non-specific staining. Do not wash.
7) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody diluted
with PBS containing 1% BSA as suggest in theAPPLICATIONS.
8) Incubate the sections for 1 hour at room temperature.
9) Wash the slides 3 times in PBS for 5 minutes each.
10) Wipe gently around each section and cover tissues withENVISION+Dual Link (Dako JAPAN; code no. 4063).
Incubate for 1 hour at room temperature. Wash as in step9).
12) Visualize by reacting for 10-20 minutes with DAB
substrate Kit (Dako JAPAN; code no. K3466). *DAB is asuspect carcinogen and must be handled with care. Always wear gloves.

13) Wash the slides in water for 5 minutes.
14) Counter stain in hematoxylin for 1 minute, wash the slides
3 times in water for 5 minutes each, and then immerse theslides in PBS for 5 minutes. Dehydrate by immersing in
Ethanol 3 times for 3 minutes each, followed byimmersing in Xylene 3 times for 3 minutes each.
15) Now ready for mounting.
(Positive control for Immunocytochemistry; HEL)

Immunohistochemical detection ofc-Kit on paraffin embedded sectionof human pancreas with 566.
Immunohistochemical staining for paraffin-embedded
sections
1) Deparaffinize the sections with Xylene 3 times for 3-5
minutes each.
2) Wash the slides with Ethanol 3 times for 3-5 minuteseach.
3) Wash the slides with PBS 3 times for 3-5 minutes each.
4) Heat treatment

566
Page 3 of 3Heat treatment by microwave oven:
Place the slides put on staining basket in 500 mL beakerwith 500 mL citrate buffer (pH 6.5). Cover the beakerwith plastic wrap, then process the slides 2 times for 10minutes each at 500 W with microwave oven. Let theslides cool down in the beaker at room temperature forabout 40 minutes.
5) Remove the slides from the citrate buffer and cover eachsection with 3% H2O2 for 10 minutes at room temperature
to block endogenous peroxidase activity. Wash 3 times inPBS for 5 minutes each.
6) Remove the slides from PBS, wipe gently around eachsection and cover tissues with 1% BSA in PBS for 5minutes to block non-specific staining. Do not wash.
7) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody dilutedwith PBS containing 1% BSA as suggest in theAPPLICATIONS.
8) Incubate the sections for 1 hour at room temperature.
9) Wash the slides 3 times in PBS for 5 minutes each.
10) Wipe gently around each section and cover tissues withENVISION+Dual Link (Dako JAPAN; code no. 4063).
Incubate for 1 hour at room temperature. Wash as in step9).
11) Visualize by reacting for 10-20 minutes with DAB
substrate Kit (Dako JAPAN; code no. K3466). *DAB is asuspect carcinogen and must be handled with care. Always wear gloves.

12) Wash the slides in water for 5 minutes.
13) Counter stain in hematoxylin for 1 minute, wash the slides
3 times in water for 5 minutes each, and then immerse theslides in PBS for 5 minutes. Dehydrate by immersing inEthanol 3 times for 3 minutes each, followed byimmersing in Xylene 3 times for 3 minutes each.
14) Now ready for mounting.
(Positive control for Immunohistochemistry; Humanpancreas)

RELATED PRODUCTS:
566-H
PM035

Anti-CD117 (c-Kit) (Human) pAb
Normal Rabbit IgG

180130-4