GPNMB Antibody (Rabbit mAb) [H14J12]

Application: Reactivity: Human

Lane 1: SK-MEL-28, Lane 2: U-87 MG, Lane 3: SKBR3, Lane 4: A375

Experiment Essentials

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

Usage Information

Dilution
WB1:500-1:1000 IF1:250 - 1:500 FCM1:500

Application
WB, IF, FCM

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
64 kDa 120 kDa,95 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	SK-MEL-28 cells; U-87 MG cells
Negative Control	HeLa cells

Experimental 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 wash the cells with ice-cold PBS twice. Lyse the cells by adding 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. Lyse the cells by adding 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min. 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 1:500), 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-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 wash the cells with ice-cold PBS twice. Lyse the cells by adding 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. Lyse the cells by adding 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.

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 1:500), 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
GPNMB Antibody (Rabbit mAb) [H14J12] detects endogenous levels of total GPNMB protein.

Subcellular Location
Cell membrane, Endosome, Membrane

Uniprot ID
Q14956

Clone
H14J12

Synonym(s)
HGFIN, NMB, UNQ1725/PRO9925, GPNMB, Transmembrane glycoprotein NMB, Hematopoietic growth factor inducible neurokinin-1 type

Background
GPNMB, also known as osteoactivin, belongs to the pMEL17/premelanosome protein family and functions as a type IA transmembrane glycoprotein with a long extracellular domain and a short intracellular domain, an architecture that reflects its dual capacity for extracellular protein-protein interactions on one side and intracellular signal propagation on the other. The extracellular domain contains an RGD motif capable of integrin engagement, a polycystic kidney disease domain that folds into an immunoglobulin-like structure, and a kringle-like domain associated with protein-binding interactions, while the cytosolic tail carries a half immunoreceptor tyrosine-based activation motif together with a dileucine motif that directs the protein toward lysosomal and endosomal compartments. GPNMB operates through several parallel mechanisms depending on cellular context, functioning as a melanosome-associated protein, a membrane-bound surface receptor, a shed soluble ligand, or an adhesion molecule, and the tyrosine residue within its intracellular hemITAM motif drives downstream signaling that supports stem-like properties and malignant progression in breast cancer cells. Proteolytic shedding releases the extracellular domain from the cell surface, and this soluble fragment interacts with integrins to recruit immune-suppressive and pro-angiogenic cell populations into the tumor microenvironment, extending GPNMB's tumor-promoting influence beyond the cell of origin through a paracrine route that runs parallel to its cell-autonomous signaling. Within the same tumor cells, GPNMB modulates receptor tyrosine kinase and integrin signaling to activate downstream kinase cascades, and this activation triggers expression and secretion of matrix metalloproteinases and cytokines that drive tumor cell proliferation, migration, and invasion, so the protein exerts its pro-tumorigenic function both intracellularly and through microenvironmental remodeling. Beyond oncology, GPNMB regulates osteoclast differentiation and activity in bone, contributes to cell differentiation, migration, and neuroprotection across multiple tissue types, and participates in inflammatory and immune-mediated processes including impairment of T-cell activation. Cerebrospinal fluid and tissue GPNMB levels rise in several neurodegenerative and neuroinflammatory conditions, and its expression is documented in immune-mediated kidney disease including lupus nephritis, positioning GPNMB as a marker of macrophage and microglial activation across autoimmune and neurological pathology in addition to its established relevance in melanoma, glioblastoma, and triple-negative breast cancer. This combination of extracellular integrin-binding motifs, a shedding-dependent paracrine mechanism, and a signaling-competent cytoplasmic tail makes GPNMB a defined target for researchers studying tumor microenvironment remodeling, osteoclast biology, and immune cell activation across disease contexts.

Background

GPNMB, also known as osteoactivin, belongs to the pMEL17/premelanosome protein family and functions as a type IA transmembrane glycoprotein with a long extracellular domain and a short intracellular domain, an architecture that reflects its dual capacity for extracellular protein-protein interactions on one side and intracellular signal propagation on the other. The extracellular domain contains an RGD motif capable of integrin engagement, a polycystic kidney disease domain that folds into an immunoglobulin-like structure, and a kringle-like domain associated with protein-binding interactions, while the cytosolic tail carries a half immunoreceptor tyrosine-based activation motif together with a dileucine motif that directs the protein toward lysosomal and endosomal compartments. GPNMB operates through several parallel mechanisms depending on cellular context, functioning as a melanosome-associated protein, a membrane-bound surface receptor, a shed soluble ligand, or an adhesion molecule, and the tyrosine residue within its intracellular hemITAM motif drives downstream signaling that supports stem-like properties and malignant progression in breast cancer cells. Proteolytic shedding releases the extracellular domain from the cell surface, and this soluble fragment interacts with integrins to recruit immune-suppressive and pro-angiogenic cell populations into the tumor microenvironment, extending GPNMB's tumor-promoting influence beyond the cell of origin through a paracrine route that runs parallel to its cell-autonomous signaling. Within the same tumor cells, GPNMB modulates receptor tyrosine kinase and integrin signaling to activate downstream kinase cascades, and this activation triggers expression and secretion of matrix metalloproteinases and cytokines that drive tumor cell proliferation, migration, and invasion, so the protein exerts its pro-tumorigenic function both intracellularly and through microenvironmental remodeling. Beyond oncology, GPNMB regulates osteoclast differentiation and activity in bone, contributes to cell differentiation, migration, and neuroprotection across multiple tissue types, and participates in inflammatory and immune-mediated processes including impairment of T-cell activation. Cerebrospinal fluid and tissue GPNMB levels rise in several neurodegenerative and neuroinflammatory conditions, and its expression is documented in immune-mediated kidney disease including lupus nephritis, positioning GPNMB as a marker of macrophage and microglial activation across autoimmune and neurological pathology in addition to its established relevance in melanoma, glioblastoma, and triple-negative breast cancer. This combination of extracellular integrin-binding motifs, a shedding-dependent paracrine mechanism, and a signaling-competent cytoplasmic tail makes GPNMB a defined target for researchers studying tumor microenvironment remodeling, osteoclast biology, and immune cell activation across disease contexts.

References
https://pubmed.ncbi.nlm.nih.gov/32445534/ https://pubmed.ncbi.nlm.nih.gov/29097143/

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%