Figure 1: Structure of IL-6 target protein.

IL-6 Target Introduction

Protein Function

• IL-6 is a cytokine with diverse biological functions in immunity, tissue regeneration, and metabolism.
• It is a potent inducer of the acute phase response. Rapid production of IL6 contributes to host defense during infection and tissue injury, but excessive IL6 synthesis is involved in disease pathology.
• It participates in lymphocyte differentiation, such as B cells and CD4+ T cells.
• It plays a role in metabolic control, and is discharged into the bloodstream after muscle contraction and acts to increase the breakdown of fats and to improve insulin resistance.

Protein Expression

• Produced by skeletal muscle. Plasma levels are highly increased upon exercise, due to enhanced production by contracting skeletal muscles.

Protein Localization

• IL-6 is a secreted protein.

Figure 2: IL-6 ICC Experiment Results
Anti-IL-6 Antibody [EPR20653] (ab214429)
Green: IL-6, Red: Tubulin, Blue: DAPI.
Treatment Conditions: Lipopolysaccharide (LPS) (0.5µg/ml for 24 hours) and Brefeldin A (BFA) (300 ng/ml for 20 hours).

Isoforms & Post-Translational Modifications

• Human (P05231): 24 kDa (predicted)
• Mouse (P08505): 24 kDa (predicted)
• Rat (P20607): 24 kDa (predicted)
• Contains N- and O-glycosylation sites.

WB Experiment Tips

Precautions

• IL-6 is typically detected in monocytes or differentiated macrophages. It is important to note that samples often require induction (e.g., LPS, IL-1 beta) to detect IL-6 (see Figure 3 and 4). Induction conditions provided in the product datasheet or literature are for reference only. For detecting IL-6 in different samples, optimize the concentration and duration of induction agents to determine the optimal conditions before formal testing. Untreated samples can serve as negative controls.
• Since IL-6 is a secretory protein, to improve the success rate of WB experiments, besides inducing sufficient expression of IL-6 in the samples, it is recommended to treat samples with BFA (Brefeldin A) after induction (see Figure 3 and 4). This inhibits the secretion of IL-6 into the extracellular space, ensuring that sufficient target protein is detectable in cell lysates.
• Maintain consistency in cell culture density to ensure the success and reproducibility of experiments. For example, with THP-1-derived macrophages, variations in cell density (too low or too high) can affect cytokine secretion, including IL-6.
• Target proteins exhibit different post-translational modifications in different samples, so the observed band sizes typically range from 21 to 28 kDa, which may differ from the predicted size of 24 kDa.
• It is advisable to include positive controls.

Positive Controls

• Human: HUVEC cell lysate treated with LPS and BFA; A549 cell lysate treated with IL-1 beta and BFA.
• Rat: NR8383 cell lysate treated with LPS and BFA.

Negative Controls

• Human: Untreated HUVEC cell lysate; untreated A549 cell lysate.
• Rat: Untreated NR8383 cell lysate.

Example Results

Figure 3: WB- Anti-IL-6 antibody [EPR21711] (ab233706)

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 2 : Wild-type A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate
Lane 3 : IL-6 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 4h) cell lysate
Lane 4 : IL-6 knockout A549 IL-1ß (ab259387) (20 ng/ml, 24h) and Brefeldin A (ab120299)-treated (5 ug/ml for the last 4h) cell lysate

Primary Antibodies:

• Anti-IL-6 antibody [EPR21711] (ab233706), at1/1000 dilution
• Anti-Alpha Tubulin antibody [DM1A] (ab7291), at1/20000 dilution

Secondary Antibodies:

• Goat anti-Rabbit IgG H&L (IRDye® 800CW) pre-adsorbed secondary antibody, at 1/20000 dilution
• Goat anti-Mouse IgG H&L (IRDye® 680RD) pre-adsorbed secondary antibody, at1/20000 dilution

Predicted Band Size: 23 kDa

Detected Band Sizes:

• Target Band: 25 kDa
• Non-specific Band: 40 kDa

Figure 4: WB- Anti-IL-6 antibody [EPR23819-11] (ab259341)

Lane 1: 20 µg of untreated NR8383 whole cell lysate
Lane 2: 20 µg of NR8383 cell lysate treated with LPS (0.1 µg/ml, 4h) and BFA (1 µg/ml, last 3h)

Primary Antibody: Anti-IL-6 antibody [EPR23819-11] (ab259341), diluted 1/1000
Secondary Antibody: Goat anti-Rabbit IgG H&L (HRP) (ab97051), diluted 1/20000
Predicted Band Size: 23 kDa
Detected Band Size: 20 kDa
Exposure time: 3 min
Result Description: The molecular weight consistent with literature descriptions (PMID: 2523818, PMID: 25130514).

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg of total protein for electrophoresis.
2. For target proteins with smaller molecular weights, it is recommended to use a 15% separating gel for electrophoresis.

Transfer:

1. It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.
2. For target proteins with a lower molecular weight, it is recommended to use a PVDF membrane with a pore size of 0.22μm.

Antibody incubation:

1. Please select a suitable antibody working concentration according to the product datasheet.

References

1. Fabrizio De Benedetti, Nadia Rucci, Andrea Del Fattore, Barbara Peruzzi, Rita Paro, Maurizio Longo, Marina Vivarelli, Flaminia Muratori, Silvia Berni, Paola Ballanti, Serge Ferrari, Anna Teti. Impaired skeletal development in interleukin-6-transgenic mice: a model for the impact of chronic inflammation on the growing skeletal system. Arthritis Rheum. 2006 Nov;54(11):3551-63. doi: 10.1002/art.22175.
2. Sujin Kang, Toshio Tanaka, Masashi Narazaki, Tadamitsu Kishimoto. Targeting Interleukin-6 Signaling in Clinic. Immunity. 2019 Apr 16;50(4):1007-1023. doi: 10.1016/j.immuni.2019.03.026.
3. Hideko Nakahara, Jian Song, Masamichi Sugimoto, Keisuke Hagihara, Tadamitsu Kishimoto, Kazuyuki Yoshizaki, Norihiro Nishimoto. Anti-interleukin-6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis. Arthritis Rheum. 2003 Jun;48(6):1521-9. doi: 10.1002/art.11143.
4. A Steensberg, G van Hall, T Osada, M Sacchetti, B Saltin, B Klarlund Pedersen. Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-induced increase in plasma interleukin-6. J Physiol. 2000 Nov 15;529 Pt 1(Pt 1):237-42. doi: 10.1111/j.1469-7793.2000.00237.x.
5. Adele V Ruder, Lieve Temmerman, Joep M A van Dommelen et al. Culture density influences the functional phenotype of human macrophages. Front Immunol. 2023 Mar 10:14:1078591. doi: 10.3389/fimmu.2023.1078591. eCollection 2023.