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GPCR RDC1/CXCR-7 Rabbit mAb

Catalog No.: F2937

Experiment Essentials

Subcellular Location: Cell membrane, Endosome, Membrane.
WB
Recommending using RIPA/NP-40 Lysis Buffer to prepare lysates.
Recommended SDS-PAGE separating gel concentration: 5%.
Recommended wet transfer conditions: 250 mA, 180 min.

Usage Information

Dilution
WB1:1000

Application
WB

Source
Rabbit

Reactivity
Human, Mouse, Rat

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
41 kDa

Exprimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 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.Add an appropriate volume of RIPA/NP-40 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: 250 mA, 180 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 5% skim milk powder 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 1389. 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.

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 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.Add an appropriate volume of RIPA/NP-40 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: 250 mA, 180 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 5% skim milk powder 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

1389. 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.

Datasheet & SDS

Biological Description

Specificity
GPCR RDC1/CXCR-7 Rabbit mAb recognizes endogenous levels of total gpcr rdc1/cxcr-7 protein.

Synonym(s)
CXCR7,GPCR RDC1

Clone
G15F11

Background
CXCR7, also known as RDC1, is an atypical G protein-coupled receptor (GPCR) that binds chemokines CXCL12 (SDF-1) and CXCL11 (I-TAC). Unlike typical GPCRs, CXCR7 does not activate classical G protein signalling pathways but instead functions as a scavenger receptor, internalizing and degrading CXCL12 through β-arrestin-mediated endocytosis. This unique mechanism regulates chemokine gradients, critical for processes like cell migration, tissue development, and immune responses. By controlling CXCL12 availability, CXCR7 modulates the activity of CXCR4, the canonical receptor for CXCL12, fine-tuning its signalling and functional responses. CXCR7 plays an essential role in embryonic development, particularly in heart morphogenesis, where it regulates semilunar valve formation and vascular remodelling. It is also crucial for immune cell trafficking, influencing the migration of B cells, T cells, and other leukocytes by shaping chemokine gradients in lymphoid organs and inflammatory sites. In cancer, CXCR7 promotes tumor progression, metastasis, and angiogenesis by altering the tumor microenvironment and enhancing the CXCR4-mediated migration of cancer cells. It also forms heterodimers with CXCR4, further modulating its signalling and impacting cell survival, proliferation, and chemotaxis. The receptor’s involvement in development and disease is mediated through its regulation of key signalling pathways, such as BMP (bone morphogenetic protein) and EGFR (epidermal growth factor receptor), which influence cell proliferation and tissue patterning. Dysregulation of CXCR7 is linked to various pathologies, including cancer, congenital heart defects, and inflammatory disorders.

Background

CXCR7, also known as RDC1, is an atypical G protein-coupled receptor (GPCR) that binds chemokines CXCL12 (SDF-1) and CXCL11 (I-TAC). Unlike typical GPCRs, CXCR7 does not activate classical G protein signalling pathways but instead functions as a scavenger receptor, internalizing and degrading CXCL12 through β-arrestin-mediated endocytosis. This unique mechanism regulates chemokine gradients, critical for processes like cell migration, tissue development, and immune responses. By controlling CXCL12 availability, CXCR7 modulates the activity of CXCR4, the canonical receptor for CXCL12, fine-tuning its signalling and functional responses. CXCR7 plays an essential role in embryonic development, particularly in heart morphogenesis, where it regulates semilunar valve formation and vascular remodelling. It is also crucial for immune cell trafficking, influencing the migration of B cells, T cells, and other leukocytes by shaping chemokine gradients in lymphoid organs and inflammatory sites. In cancer, CXCR7 promotes tumor progression, metastasis, and angiogenesis by altering the tumor microenvironment and enhancing the CXCR4-mediated migration of cancer cells. It also forms heterodimers with CXCR4, further modulating its signalling and impacting cell survival, proliferation, and chemotaxis. The receptor’s involvement in development and disease is mediated through its regulation of key signalling pathways, such as BMP (bone morphogenetic protein) and EGFR (epidermal growth factor receptor), which influence cell proliferation and tissue patterning. Dysregulation of CXCR7 is linked to various pathologies, including cancer, congenital heart defects, and inflammatory disorders.

References
[1] Yu S, et al. Dev Dyn. 2011 Feb;240(2):384-93. [2] Levoye A, et al. Blood. 2009 Jun 11;113(24):6085-93.

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
If you have any other enquiries, please leave a message.

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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%