The total protein was extracted from samples of gastric biopsies using RIPA buffer containing inhibitors (1 M NaF, Complete Protease Cocktail Inhibitor, and 0.1 M PMSF). The total extracted proteins were quantified using the NanoDrop 2000 spectrophotometer (Thermo Scientific®) using Bradford reagent and standard BSA proteins (5000201, Bio-Rad, USA). The proteins (25 μg) were separated in 12% SDS-PAGE, and transferred to a nitrocellulose membrane, and then blocked with 5% skimmed milk in 1× phosphate buffered saline and 0.1% Tween 20 (PBST), for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C, with primary antibodies: anti-α-tubulin antibody (5168, 1:5,000, Santa Cruz Biotechnology, USA); anti-IL-6 (93356, 1:500, Abcam, USA), and anti-IL-6R (128008, 1:3,000, Abcam). Then, the membranes were incubated individually for 1 h 30 min at room temperature with secondary antibodies: goat anti-rabbit IgG H&L (97051, 1:10,000, Abcam), used for IL-6 and IL-6R, and m-IgGk BP-HRP (1:5.000 Santa Cruz Biotechnology), used for tubulin. Finally, the membranes were washed in PBST, and revealed.
Anti il 6r
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-IL-6R is a laboratory reagent used in scientific research. It specifically binds to the interleukin-6 receptor (IL-6R), a cell surface protein involved in immune and inflammatory responses. The product can be used to study the function and regulation of IL-6R in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 1r, by Abcam (2 mentions)
Goat anti rabbit igg h l, by Abcam (1 mentions)
Anti α tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Anti il 6, by Abcam (1 mentions)
M iggk bp hrp, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Igf1r, by Merck Group (1 mentions)
Ecl method, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti il 6, by Santa Cruz Biotechnology (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase, by Proteintech (1 mentions)
Anti stat3, by Santa Cruz Biotechnology (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Merck Group (1 mentions)
Anti bax, by Merck Group (1 mentions)
G box imaging system, by Syngene (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
7 protocols using anti il 6r
1
Gastric Biopsy Protein Extraction and Analysis
2
Western Blot Analysis of IL-6R Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain the total proteins, RIPA lysis buffer added with PMSF was used at 4°C. The supernatant was obtained after cells were centrifuged at 9500 g/min for 5 minutes. Afterwards, BCA Protein Assay Kit (Beyotime, Shanghai, China) was carried out to measure protein concentration and the protein was boiled for degeneration. Then, the proteins were loaded on 10% SDS‐PAGE and transferred to PVDF membranes. We added the primary antibodies to the membranes for a whole night at 4°C. Primary antibodies, anti‐IL‐6R and anti‐GAPDH (1:1000, Abcam, Cambridge, UK), were used. After incubated with secondary antibodies for 2 hours, the membranes were exposed to the enhanced chemiluminescence reagent. Finally, the bands were evaluated utilizing ImageJ software.
3
Protein Detection in Cell Lysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer. The proteins were detected with anti-IL-6 (Santa Cruz, Dallas, TX, USA, sc-7920), anti-IL-6R (Abcam, Cambridge, UK, ab128008), IGF1R (Sigma-Aldrich, St Louis, MO, USA, SAB1104892), anti-STAT3 (Santa Cruz, sc-482), anti-phoshpo-STAT3 (Cell Signaling, Danvers, MA, USA, 9145), anti-phoshpo-IGF1R (Cell Signaling, 2969), anti-beta-actin (Cell Signaling, 4970) or anti-glyceraldehyde 3-phosphate dehydrogenase (Protein Tech Group, Chicago, IL, USA, 10494-1-AP). Immunoreactivity was determined using the ECL method (Millipore, Boston, MA, USA) according to the manufacturer's instructions.
4
Protein Expression Analysis in Neurons and Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extracted from primary neurons or astrocytes was used for western blotting. The membranes were incubated in primary antibodies including anti-IL6R (1:400, Abcam, USA), anti-STAT3 (1:800, ABZOOM, USA), anti-Bax (1:200, Sigma, USA), anti-Bcl-2 (1:800, Sigma, USA) and anti-β-actin (1:500, Santa Cruz, USA) at 4 °C overnight, followed by incubation in HRP-conjugated secondary antibodies (Santa Cruz, USA) at room temperature for 1 h. Then, the protein bands were detected using a chemiluminescent HRP substrate (Millipore, USA). The images were captured using a Syngene GBox Imaging System.
5
Western Blot Analysis of IL-6R Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole protein extracts were prepared and analyzed by Western blotting, according to a standard procedure. The following antibodies were used: anti-IL-6R (Abcam, Cambridge, UK); anti-phospho STAT3 (Tyr705, clone 3E2) and anti-STAT3 (Cell Signaling Technology, Danvers, MA, USA); anti-α-tubulin clone DM1A (Sigma-Aldrich, Milan, Italy). Densitometric analysis was performed using the ImageJ software. The original Western blotting images are shown in Figure S3 .
6
Western Blot Analysis of Immune Receptors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The western blot was performed as described previously.40 Briefly, NPs, MMNPs, macrophage membrane extraction, or total protein extracted from heart tissues were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non‐fat milk for overnight at 4°C. Next day, primary antibodies were added to the membrane and incubated at room temperature for 2 h. After wash, corresponding horse radish peroxidase‐conjugated secondary antibodies were incubated for 1 h at room temperature. Immuno‐reactive bands were visualized using ECL substrate kit (Abcam, China). Antibodies used in present study include: anti‐IL‐1R (Abcam, Beijing, China), anti‐IL‐6R (Abcam, China), anti‐tumor necrosis factor receptor 1 (TNFRI) (Abcam, China), anti‐CD86 (Abcam, China), and anti‐CD206 (Abcam, China).
7
Quantifying Inflammatory Receptor Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After being denatured by heating at 95°C in an SDS sample buffer, the
supernatant samples were loaded onto Mini-Protean TGX Precast gels (Bio-Rad
Laboratories, Hercules, CA, USA) and electrically transferred to a polyvinylidene
fluoride membrane. The membrane was then incubated overnight with primary antibodies:
rabbit anti-IL-1R, anti-IL-6R and anti-TNFR1 (Abcam Co., Cambridge, UK, diluted
1:200). After being fully washed, the membrane was incubated with horseradish
peroxidase-linked anti-rabbit secondary antibody and visualized for immunoreactivity.
The membrane was stripped and incubated with mouse anti-β-actin to show equal
loading of the protein. The bands recognized by the primary antibody were visualized
by exposure of the membrane onto an X-ray film. Then, the film was scanned and the
optical densities of IL-1R, IL-6R, TNFR1 and β-actin bands were determined
using the public domain ImageJ program (made by Wayne Rasband, Scion Corporation,
Bethesda, MD, USA). Then, values for densities of immunoreactive bands/β-actin
band from the same lane were determined. Each of the values was then normalized to a
control sample.
supernatant samples were loaded onto Mini-Protean TGX Precast gels (Bio-Rad
Laboratories, Hercules, CA, USA) and electrically transferred to a polyvinylidene
fluoride membrane. The membrane was then incubated overnight with primary antibodies:
rabbit anti-IL-1R, anti-IL-6R and anti-TNFR1 (Abcam Co., Cambridge, UK, diluted
1:200). After being fully washed, the membrane was incubated with horseradish
peroxidase-linked anti-rabbit secondary antibody and visualized for immunoreactivity.
The membrane was stripped and incubated with mouse anti-β-actin to show equal
loading of the protein. The bands recognized by the primary antibody were visualized
by exposure of the membrane onto an X-ray film. Then, the film was scanned and the
optical densities of IL-1R, IL-6R, TNFR1 and β-actin bands were determined
using the public domain ImageJ program (made by Wayne Rasband, Scion Corporation,
Bethesda, MD, USA). Then, values for densities of immunoreactive bands/β-actin
band from the same lane were determined. Each of the values was then normalized to a
control sample.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!