ROR2 Antibody (Rabbit mAb) [A21B16]

Application: Reactivity: Human

Lane 1: HeLa, Lane 2: HepG2

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IHC1:500

Application
WB, IHC

Reactivity
Human

Source
Rabbit Monoclonal Antibody

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

Predicted MW Observed MW
105 kDa 105-130 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	HAP1 cells; HeLa cells; HepG2 cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-42 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-42 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-42 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-42 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-42 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-42 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

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
ROR2 Antibody (Rabbit mAb) [A21B16] detects endogenous levels of total ROR2 protein.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
Q01974

Clone
A21B16

Synonym(s)
NTRKR2, ROR2, Tyrosine-protein kinase transmembrane receptor ROR2

Background
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a single-pass transmembrane receptor of the Ror family that carries extracellular immunoglobulin-like, cysteine-rich and Kringle domains and an intracellular tyrosine kinase domain, and it acts as a receptor or coreceptor for Wnt5a to mediate noncanonical Wnt signaling. Wnt5a binds directly to the cysteine-rich domain of ROR2, and this interaction can occur in the context of a receptor complex where ROR2 associates with Frizzled family members such as Frizzled2 or Frizzled7 via its extracellular region, forming a Wnt5a-responsive unit that regulates downstream signaling components including Dishevelled. Wnt5a stimulation induces ROR2 homodimerization and tyrosine phosphorylation in osteoblastic cells and increases phosphorylation of the scaffold protein 14‑3‑3β, demonstrating activation of a classical receptor tyrosine kinase cascade that includes direct substrate phosphorylation by ROR2 and contributes to osteoblast differentiation and bone formation. Genetic studies show that Ror2‑/‑ and Wnt5a‑/‑ mice share developmental defects including dwarfism, limb and facial abnormalities, lung and genital dysplasia and ventricular septal defects, and Wnt5a–ROR2 signaling inhibits convergent extension movements in Xenopus embryos while activating c‑Jun N‑terminal kinase, placing ROR2 in a Wnt5a‑dependent noncanonical Wnt/JNK pathway that controls morphogenetic cell movements. In hematopoietic K562 cells, Wnt5a increases ROR2 expression and induces its internalization and co-localization with Wnt5a, while ROR2 binding to Wnt5a interferes with Wnt5a association with Frizzled4 and LRP5, shifting signaling away from the β‑catenin‑dependent canonical pathway. Under these conditions, Wnt5a–ROR2 signaling regulates tyrosine phosphorylation and nuclear translocation of β‑catenin and suppresses β‑catenin/TCF-dependent transcriptional activity, with down-regulation of cyclin D1 expression, identifying a mechanism in which the Wnt5a/ROR2 noncanonical axis inhibits canonical Wnt signaling and exerts tumor-suppressive effects in leukemic models. ROR2/Fz receptor complexes also mediate Wnt5a-induced AP‑1 activation by regulating Dishevelled polymerization; ROR2 associates with Frizzled7 via its cysteine-rich domain, this complex promotes Wnt5a-triggered Dishevelled polymerization that requires both DIX and DEP domains, and polymerized Dishevelled co-localizes with Rac1 whose activity is necessary for AP‑1 promoter activation, delineating a Wnt5a–ROR2–Frizzled7–Dishevelled–Rac1–AP‑1 signaling axis. ROR2 acts as a Wnt5a-binding receptor tyrosine kinase that integrates extracellular Wnt5a gradients into noncanonical signaling outputs involving JNK, Rac1 and AP‑1, while modulating β‑catenin activity and cyclin D1 expression, and its roles in skeletal morphogenesis, osteoblast differentiation and regulation of canonical Wnt signaling underpin its relevance as a target in developmental biology and in tumor contexts where Wnt5a/ROR2 signaling contributes to migration, invasion or suppression of β‑catenin-driven transcription.

Background

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a single-pass transmembrane receptor of the Ror family that carries extracellular immunoglobulin-like, cysteine-rich and Kringle domains and an intracellular tyrosine kinase domain, and it acts as a receptor or coreceptor for Wnt5a to mediate noncanonical Wnt signaling. Wnt5a binds directly to the cysteine-rich domain of ROR2, and this interaction can occur in the context of a receptor complex where ROR2 associates with Frizzled family members such as Frizzled2 or Frizzled7 via its extracellular region, forming a Wnt5a-responsive unit that regulates downstream signaling components including Dishevelled. Wnt5a stimulation induces ROR2 homodimerization and tyrosine phosphorylation in osteoblastic cells and increases phosphorylation of the scaffold protein 14‑3‑3β, demonstrating activation of a classical receptor tyrosine kinase cascade that includes direct substrate phosphorylation by ROR2 and contributes to osteoblast differentiation and bone formation. Genetic studies show that Ror2‑/‑ and Wnt5a‑/‑ mice share developmental defects including dwarfism, limb and facial abnormalities, lung and genital dysplasia and ventricular septal defects, and Wnt5a–ROR2 signaling inhibits convergent extension movements in Xenopus embryos while activating c‑Jun N‑terminal kinase, placing ROR2 in a Wnt5a‑dependent noncanonical Wnt/JNK pathway that controls morphogenetic cell movements. In hematopoietic K562 cells, Wnt5a increases ROR2 expression and induces its internalization and co-localization with Wnt5a, while ROR2 binding to Wnt5a interferes with Wnt5a association with Frizzled4 and LRP5, shifting signaling away from the β‑catenin‑dependent canonical pathway. Under these conditions, Wnt5a–ROR2 signaling regulates tyrosine phosphorylation and nuclear translocation of β‑catenin and suppresses β‑catenin/TCF-dependent transcriptional activity, with down-regulation of cyclin D1 expression, identifying a mechanism in which the Wnt5a/ROR2 noncanonical axis inhibits canonical Wnt signaling and exerts tumor-suppressive effects in leukemic models. ROR2/Fz receptor complexes also mediate Wnt5a-induced AP‑1 activation by regulating Dishevelled polymerization; ROR2 associates with Frizzled7 via its cysteine-rich domain, this complex promotes Wnt5a-triggered Dishevelled polymerization that requires both DIX and DEP domains, and polymerized Dishevelled co-localizes with Rac1 whose activity is necessary for AP‑1 promoter activation, delineating a Wnt5a–ROR2–Frizzled7–Dishevelled–Rac1–AP‑1 signaling axis. ROR2 acts as a Wnt5a-binding receptor tyrosine kinase that integrates extracellular Wnt5a gradients into noncanonical signaling outputs involving JNK, Rac1 and AP‑1, while modulating β‑catenin activity and cyclin D1 expression, and its roles in skeletal morphogenesis, osteoblast differentiation and regulation of canonical Wnt signaling underpin its relevance as a target in developmental biology and in tumor contexts where Wnt5a/ROR2 signaling contributes to migration, invasion or suppression of β‑catenin-driven transcription.

References
https://pubmed.ncbi.nlm.nih.gov/12839624/ https://pubmed.ncbi.nlm.nih.gov/18615587/

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

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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%