CCL3/MIP-1α Antibody (Rabbit mAb) [C14N6]

Application: Reactivity: Mouse

Lane 1: RAW 264.7, Lane 2: RAW 264.7 (LPS, 100ng/mL; BFA, 300ng/ml, 4 h)

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 20%.
Recommended wet transfer conditions: 200 mA, 60 min，Recommended to use 0.22 μm PVDF membrane.
Exposure time of at least 60s is recommended.

Usage Information

Dilution
WB1:1000 IP1:30 IF1:500 FCM1:600

Application
WB, IP, IF, FCM

Reactivity
Mouse

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
10 kDa 10 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	Recombinant mouse Macrophage Inflammatory Protein 1 alpha / CCL3 protein; RAW 264.7 cells (LPS, 100 ng/ml, 3 h; Brefeldin A, 300 ng/ml, added for the last 3 h)
Negative Control	RAW 264.7 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. 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: 20%. 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. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 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. (Exposure time of at least 60s is recommended)

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. 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: 20%. 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. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 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. (Exposure time of at least 60s is recommended)

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. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) 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.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

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
CCL3/MIP-1α Antibody (Rabbit mAb) [C14N6] detects endogenous levels of total CCL3/MIP-1α protein.

Subcellular Location
Secreted

Uniprot ID
P10147

Clone
C14N6

Synonym(s)
Mip1a \| Scya3 \| Ccl3 \| C-C motif chemokine 3 \| Heparin-binding chemotaxis protein \| L2G25B \| Macrophage inflammatory protein 1-alpha \| SIS-alpha \| Small-inducible cytokine A3 \| TY-5 \| MIP-1-alpha

Background
CCL3, also known as macrophage inflammatory protein-1α (MIP-1α), is a CC chemokine produced mainly by activated macrophages, T cells and other myeloid-lineage cells that functions as a potent chemoattractant and activation signal for leukocytes, integrating inflammatory cues with chemokine receptor signaling to shape innate and adaptive immune responses. The protein belongs to the CC chemokine family and adopts the typical compact chemokine fold with an N‑terminal region that engages G protein–coupled receptors and a conserved cysteine motif that stabilizes its structure, allowing high-affinity binding to CCR1 and CCR5 on target cells. Upon binding to CCR1 or CCR5, CCL3 triggers Gαi‑coupled signaling that activates downstream ERK1/2, JNK and p38 MAPK pathways, increases intracellular calcium and reorganizes the actin cytoskeleton, resulting in directional chemotaxis of monocytes, neutrophils, NK cells and subsets of T lymphocytes toward inflammatory sites. In GM‑CSF–primed neutrophils, short-term exposure induces CCR1 and CCR5 expression and renders neutrophils responsive to CCL3, where CCR5-dependent ERK1/2 activation drives in vitro migration and acquisition of locomotory behavior, demonstrating a context in which priming cytokines license CCL3 responsiveness through receptor and MAPK pathway modulation. CCL3 also upregulates CCR1/CCR5 and activates JNK/p38 MAPK signaling in pancreatic acinar cells during acute pancreatitis, leading to increased TNF‑α and IL‑6 production, reinforcing inflammatory cascades and supporting its role as both chemotactic and a proinflammatory effector. In rheumatoid arthritis, CCL3 is highly expressed in synovial tissue and fluid, where it promotes leukocyte recruitment into the joint, enhances secretion of other inflammatory mediators, supports angiogenesis and contributes to bone and cartilage destruction, positioning CCL3 as a key mediator of joint inflammation and structural damage. CCL3 also acts beyond classical inflammation: in the hippocampus, experimental elevation of CCL3 impairs basal synaptic transmission at Schaffer collateral–CA1 synapses and selectively disrupts long-term potentiation without affecting NMDA receptor potentials, effects reversed by the CCR5 antagonist maraviroc, indicating that CCL3–CCR5 signaling serves as a neuromodulatory mechanism that can negatively regulate synaptic plasticity and memory. In HIV infection, CCL3 is one of the natural ligands for CCR5, the major coreceptor used by R5-tropic HIV‑1; its binding to CCR5 competitively blocks gp120 engagement and inhibits viral entry into CD4+ T cells, and gp120-specific CD4+ T cells that secrete CCR5 ligands, including CCL3, can suppress HIV infection of autologous cells, illustrating a protective facet of CCL3–CCR5 interactions in antiviral immunity. However, serum CCL3 levels do not correlate reliably with HIV disease stage or treatment response, so its systemic concentration is not a robust biomarker even though local CCR5 ligand production is mechanistically important for viral restriction. In B-cell malignancies such as chronic lymphocytic leukemia, B‑cell receptor engagement induces CCL3 secretion by CLL cells, and elevated plasma CCL3 associates with more aggressive disease and enhanced microenvironmental interactions, making it a useful surrogate for ongoing BCR signaling and a candidate marker for disease progression.

Background

CCL3, also known as macrophage inflammatory protein-1α (MIP-1α), is a CC chemokine produced mainly by activated macrophages, T cells and other myeloid-lineage cells that functions as a potent chemoattractant and activation signal for leukocytes, integrating inflammatory cues with chemokine receptor signaling to shape innate and adaptive immune responses. The protein belongs to the CC chemokine family and adopts the typical compact chemokine fold with an N‑terminal region that engages G protein–coupled receptors and a conserved cysteine motif that stabilizes its structure, allowing high-affinity binding to CCR1 and CCR5 on target cells. Upon binding to CCR1 or CCR5, CCL3 triggers Gαi‑coupled signaling that activates downstream ERK1/2, JNK and p38 MAPK pathways, increases intracellular calcium and reorganizes the actin cytoskeleton, resulting in directional chemotaxis of monocytes, neutrophils, NK cells and subsets of T lymphocytes toward inflammatory sites. In GM‑CSF–primed neutrophils, short-term exposure induces CCR1 and CCR5 expression and renders neutrophils responsive to CCL3, where CCR5-dependent ERK1/2 activation drives in vitro migration and acquisition of locomotory behavior, demonstrating a context in which priming cytokines license CCL3 responsiveness through receptor and MAPK pathway modulation. CCL3 also upregulates CCR1/CCR5 and activates JNK/p38 MAPK signaling in pancreatic acinar cells during acute pancreatitis, leading to increased TNF‑α and IL‑6 production, reinforcing inflammatory cascades and supporting its role as both chemotactic and a proinflammatory effector. In rheumatoid arthritis, CCL3 is highly expressed in synovial tissue and fluid, where it promotes leukocyte recruitment into the joint, enhances secretion of other inflammatory mediators, supports angiogenesis and contributes to bone and cartilage destruction, positioning CCL3 as a key mediator of joint inflammation and structural damage. CCL3 also acts beyond classical inflammation: in the hippocampus, experimental elevation of CCL3 impairs basal synaptic transmission at Schaffer collateral–CA1 synapses and selectively disrupts long-term potentiation without affecting NMDA receptor potentials, effects reversed by the CCR5 antagonist maraviroc, indicating that CCL3–CCR5 signaling serves as a neuromodulatory mechanism that can negatively regulate synaptic plasticity and memory. In HIV infection, CCL3 is one of the natural ligands for CCR5, the major coreceptor used by R5-tropic HIV‑1; its binding to CCR5 competitively blocks gp120 engagement and inhibits viral entry into CD4+ T cells, and gp120-specific CD4+ T cells that secrete CCR5 ligands, including CCL3, can suppress HIV infection of autologous cells, illustrating a protective facet of CCL3–CCR5 interactions in antiviral immunity. However, serum CCL3 levels do not correlate reliably with HIV disease stage or treatment response, so its systemic concentration is not a robust biomarker even though local CCR5 ligand production is mechanistically important for viral restriction. In B-cell malignancies such as chronic lymphocytic leukemia, B‑cell receptor engagement induces CCL3 secretion by CLL cells, and elevated plasma CCL3 associates with more aggressive disease and enhanced microenvironmental interactions, making it a useful surrogate for ongoing BCR signaling and a candidate marker for disease progression.

References
https://pubmed.ncbi.nlm.nih.gov/37227653/ https://pmc.ncbi.nlm.nih.gov/articles/PMC7124056/

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%