Retinas were collected as described previously.38 (link) Equal amounts of protein from cell lysates or retinal homogenates were combined with Laemmli buffer, boiled for 5 min, and fractionated using a 4-20% Criterion TGX Precast gel (Bio-Rad Laboratories). Proteins were transferred to a polyvinylidene fluorine membrane, blocked using 5% milk in Tris-buffered saline Tween 20, and incubated with antibodies (Supplementary Table S1 ). The concentration of IL-1β was determined in the retina whole cell lysates or in culture medium using the DuoSet mouse (DY401-05) and human (DY201-05) IL-1β/IL-1F2 ELISA kits (R&D Systems, Minneapolis, MN, USA), respectively.
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Retinal Protein Isolation and IL-1β Quantification
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Corresponding Organization : Penn State Milton S. Hershey Medical Center
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Variable analysis
independent variables
- Retinal homogenates
- Cell lysates
- Protein expression
- IL-1β concentration in retina whole cell lysates
- IL-1β concentration in culture medium
- Equal amounts of protein from cell lysates or retinal homogenates
- Laemmli buffer
- 4-20% Criterion TGX Precast gel (Bio-Rad Laboratories)
- Polyvinylidene fluorine membrane
- 5% milk in Tris-buffered saline Tween 20
- Antibodies (as listed in Supplementary Table S1)
- DuoSet mouse (DY401-05) and human (DY201-05) IL-1β/IL-1F2 ELISA kits (R&D Systems, Minneapolis, MN, USA)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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