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Anti-RANK Mouse Antibody [E4N12]

Catalog No.: F2425

Application: Reactivity: Human, Mouse

Immunohistochemical analysis of formalin fixed paraffin embedded human gastric cancer tissue with F2425 at 1:50 dilution.,
Immunohistochemical analysis of formalin fixed paraffin embedded human gastric cancer tissue with F2425 at 1:50 dilution.,

Usage Information

Dilution
IP1:500-1:1000 IHC1:200

Application
IP, IHC

Reactivity
Human, Mouse

Source
Mouse

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Positive Control	Human skin; Human t cell lymphoma; RAW cell
Negative Control

Exprimental Methods

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
Anti-RANK Mouse Antibody [E4N12] detects endogenous levels of total RANK protein.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
Q9Y6Q6

Clone
E4N12

Synonym(s)
CD265, RANK, TNFRSF11A, Tumor necrosis factor receptor superfamily member 11A, Osteoclast differentiation factor receptor, Receptor activator of NF-KB, ODFR

Background
RANK (Receptor Activator of Nuclear Factor κB) is a type I transmembrane glycoprotein of 616 amino acids, comprising a 28-amino-acid signal peptide, a 184-amino-acid extracellular domain, a 21-amino-acid transmembrane region, and a 383-amino-acid intracellular domain containing three functional cytoplasmic motifs—PFQEP(369–373), PVQEET(559–564), and PVQEQG(604–609)—that mediate interactions with adaptor proteins such as TRAF6 to activate NF-κB, MAPK (JNK, ERK, p38), and Akt/PKB pathways. Encoded by the TNFRSF11A gene, RANK is expressed in osteoclast precursors, dendritic cells, monocytes, macrophages, activated T cells, natural killer cells, thymic medullary epithelial cells, mammary epithelial cells, and certain epithelial stem cell populations. Functionally, RANK regulates osteoclast differentiation, activation, and survival in bone remodeling; supports lymph node organogenesis, thymic epithelial development, and immune tolerance; maintains epithelial stem cell homeostasis; mediates progesterone-driven mammary epithelial proliferation; and, when dysregulated, contributes to tumorigenesis, metastasis, and cancer stem cell expansion.

Background

RANK (Receptor Activator of Nuclear Factor κB) is a type I transmembrane glycoprotein of 616 amino acids, comprising a 28-amino-acid signal peptide, a 184-amino-acid extracellular domain, a 21-amino-acid transmembrane region, and a 383-amino-acid intracellular domain containing three functional cytoplasmic motifs—PFQEP(369–373), PVQEET(559–564), and PVQEQG(604–609)—that mediate interactions with adaptor proteins such as TRAF6 to activate NF-κB, MAPK (JNK, ERK, p38), and Akt/PKB pathways. Encoded by the TNFRSF11A gene, RANK is expressed in osteoclast precursors, dendritic cells, monocytes, macrophages, activated T cells, natural killer cells, thymic medullary epithelial cells, mammary epithelial cells, and certain epithelial stem cell populations. Functionally, RANK regulates osteoclast differentiation, activation, and survival in bone remodeling; supports lymph node organogenesis, thymic epithelial development, and immune tolerance; maintains epithelial stem cell homeostasis; mediates progesterone-driven mammary epithelial proliferation; and, when dysregulated, contributes to tumorigenesis, metastasis, and cancer stem cell expansion.

References
https://pubmed.ncbi.nlm.nih.gov/33552088/ https://pubmed.ncbi.nlm.nih.gov/26749530/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%