IL-6 Antibody [L17N21] | Primary Antibodies

Application: Reactivity: Human

Lane 1: Recombinant Human Interleukin-6

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 IP1:100

Application
WB, IP

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
21-28 kDa

Positive Control	Recombinant Human Interleukin-6 (hIL-6); NK-92 cells (treated with TPA, ionomycin, BFA); NCI-H460 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-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 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) and put the sample on ice for 5 min. 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) and put the sample on ice for 5 min. 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: 10%. 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, 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 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 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.

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) and put the sample on ice for 5 min.

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) and put the sample on ice for 5 min.

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: 10%. 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, 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 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

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.

Datasheet & SDS

Biological Description

Specificity
IL-6 Antibody [L17N21] detects endogenous levels of total IL-6 protein.

Subcellular Location
Secreted

Uniprot ID
P05231

Clone
L17N21

Synonym(s)
B cell stimulatory factor-2; B-cell differentiation factor; B-cell stimulatory factor 2; BSF-2; BSF2; CDF; HGF; HSF; Hybridoma growth factor; interleukin BSF-2; Interleukin-6

Background
Interleukin-6 (IL-6) belongs to the IL-6 family of cytokines that share gp130 as a common signal-transducing receptor subunit. The protein adopts a four-helix bundle with up-up-down-down topology, where helices A-D connect through loops, including a mini-helix in the CD loop; 157 of 185 residues order precisely, while the N-terminus (residues 1-18) and A-B loop portion remain disordered. IL-6 engages IL-6Rα first, whose extracellular region spans three domains: N-terminal Ig-like D1 (residues 1–93) disulfide-bonded (C6-C174 in D2), cytokine-binding D2 (residues 94–194), and D3 (residues 195–299) forming fibronectin type III modules with WSXWS motif (residues 284–287). Binding occurs at D2-D3 junction via residues P162, E163 (L3), S228, F229, Y230, R231 (L5), E278, F279 (L7), enabling head-to-tail receptor dimerization through hydrophobic contacts like F134, F168. This IL-6/IL-6Rα heterodimer recruits gp130, where its membrane-distal Ig-like D1 and cytokine-binding D2-D3 domains interact with IL-6 sites II and III, forming a 2:2:2 hexameric complex that dimerises gp130 for signalling. Produced by T cells, macrophages, fibroblasts, and endothelial cells, IL-6 initiates acute phase responses by prompting hepatocytes to generate C-reactive protein and acute phase proteins. IL-6 promotes B cell differentiation into plasma cells, thymocyte and T cell proliferation, and Th17 development with TGF-β.[user-provided] Gp130 dimerization activates JAK/STAT (primarily STAT3), SHP-2/ERK MAPK, and PI3K pathways, driving gene expression for inflammation, hematopoiesis, and immune regulation. Soluble IL-6Rα facilitates trans-signalling on gp130-positive cells that lack membrane IL-6Rα, thereby amplifying its actions on endothelial cells, fibroblasts, and smooth muscle in inflammatory conditions. Elevated IL-6 fosters tumor growth, VEGF-mediated angiogenesis, and metastasis, notably in breast cancer. Dysregulated signaling underlies rheumatoid arthritis, multiple myeloma, prostate cancer, postmenopausal osteoporosis, and cardiovascular inflammation.

Background

Interleukin-6 (IL-6) belongs to the IL-6 family of cytokines that share gp130 as a common signal-transducing receptor subunit. The protein adopts a four-helix bundle with up-up-down-down topology, where helices A-D connect through loops, including a mini-helix in the CD loop; 157 of 185 residues order precisely, while the N-terminus (residues 1-18) and A-B loop portion remain disordered. IL-6 engages IL-6Rα first, whose extracellular region spans three domains: N-terminal Ig-like D1 (residues 1–93) disulfide-bonded (C6-C174 in D2), cytokine-binding D2 (residues 94–194), and D3 (residues 195–299) forming fibronectin type III modules with WSXWS motif (residues 284–287). Binding occurs at D2-D3 junction via residues P162, E163 (L3), S228, F229, Y230, R231 (L5), E278, F279 (L7), enabling head-to-tail receptor dimerization through hydrophobic contacts like F134, F168. This IL-6/IL-6Rα heterodimer recruits gp130, where its membrane-distal Ig-like D1 and cytokine-binding D2-D3 domains interact with IL-6 sites II and III, forming a 2:2:2 hexameric complex that dimerises gp130 for signalling. Produced by T cells, macrophages, fibroblasts, and endothelial cells, IL-6 initiates acute phase responses by prompting hepatocytes to generate C-reactive protein and acute phase proteins. IL-6 promotes B cell differentiation into plasma cells, thymocyte and T cell proliferation, and Th17 development with TGF-β.[user-provided] Gp130 dimerization activates JAK/STAT (primarily STAT3), SHP-2/ERK MAPK, and PI3K pathways, driving gene expression for inflammation, hematopoiesis, and immune regulation. Soluble IL-6Rα facilitates trans-signalling on gp130-positive cells that lack membrane IL-6Rα, thereby amplifying its actions on endothelial cells, fibroblasts, and smooth muscle in inflammatory conditions. Elevated IL-6 fosters tumor growth, VEGF-mediated angiogenesis, and metastasis, notably in breast cancer. Dysregulated signaling underlies rheumatoid arthritis, multiple myeloma, prostate cancer, postmenopausal osteoporosis, and cardiovascular inflammation.

References
https://pubmed.ncbi.nlm.nih.gov/32765502/ https://pubmed.ncbi.nlm.nih.gov/12461182/

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%