Mouse corneal samples were lysed with RIPA buffer. The lysates were centrifuged to obtain the supernatant. Protein concentration was determined by BCA assay. The protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes (Bio-Rad; Hercules, CA, USA). The membranes were blocked with 3% BSA and subsequently incubated with primary and secondary antibodies. Signals were visualized using SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Pittsburgh, PA, USA) using a Kodak Imaging Station 4000R. β-Actin was used as the loading control. Antibodies: anti-human IL36 α, γ and RA (α, 1 μg/ml; γ, 0.5 μg/ml; RA (0.1 μg/ml, R&DR&D) anti-mouse IL36Ra IL1Ra (1.0 μg/ml) or IL1Ra (0.1 μg/ml, Thermo Fisher Scientific), rabbit anti-mouse IL36γ was generated in Dr. Standiford lab, University of Michigan (29 (link)); and anti-β-actin (A1978; Sigma, 1:10000).
Protocol detail
Western Blot Analysis of Mouse Corneal IL-36 Proteins
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nitrocellulose membranes
SuperSignal® West Pico Chemiluminescent Substrate
Imaging Station 4000R
IL1Ra
anti-β-actin
Actin
Antibodies
Assay
Buffer
Corneal
Electrically
Human
Il1ra
Membranes
Mouse
Nitrocellulose
Protein
Rabbit
Ripa
Sds page
Corresponding Organization :
Other organizations : Wayne State University, Qilu Hospital of Shandong University
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Variable analysis
independent variables
- Protein samples
- Protein concentration
- Protein expression levels of IL36α, IL36γ, IL36RA, and IL1RA
- β-Actin as the loading control
- No positive or negative controls were explicitly mentioned.
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