Adult retinas were lysed in RIPA buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, protease inhibitors (Complete; Mini Protease Inhibitor Cocktail Tablets; Roche)] containing phosphatase inhibitors. Proteins were analysed by SDS-PAGE and transferred onto nitrocellulose membranes, which were blocked with 5% non-fat dry milk in 1 × PBS containing 0.1%Tween 20 and incubated overnight at 4 °C with primary antibodies. After incubation with horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature, immunodetection was developed using the ECL system (Lumi-Light Western Blotting Substrate, Roche). Images were acquired by ImageQuant™ LAS 4000 mini Image Analyzer (Fuji-film) and quantified using ImageJ software. α-TUBULIN or GAPDH loading controls were used to normalize protein values. The primary antibodies were the following: GSR (1:1000, 18257-1-AP, Proteintech), GPX4 (1:1000, 52455, Cell Signaling Technology), Caspase-7 p11 (1:1000, PA5-90312, Thermo Fisher Scientific), FTH1 (1:1000, 4393, Cell Signaling Technology), KEAP1 (1:1000, 8047, Cell Signaling Technology), TUBULIN (1:1000, T5168, Sigma), GAPDH (1:1000, ab8245, Abcam). The secondary antibodies were: HRP-labeled anti-mouse (1:2000, A5906, Sigma) and anti-rabbit (1:2000, NA934-100UL, GE Healthcare).
Na934 100ul
Manufactured by GE Healthcare
Sourced in United States
The NA934-100UL is a laboratory equipment product. It is designed to perform specific functions within a laboratory setting. The product details and intended use are not available in a concise, unbiased, and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Mini protease inhibitor cocktail tablet, by Roche (1 mentions)
Imagequant las 4000 mini image analyzer, by Fujifilm (1 mentions)
T5168, by Merck Group (1 mentions)
Lumi light western blotting substrate, by Roche (1 mentions)
Ab8245, by Abcam (1 mentions)
Na931 100ul, by GE Healthcare (1 mentions)
Pierce 660 nm protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
2 protocols using na934 100ul
1
Western Blot Analysis of Retinal Proteins
2
Western Blot Analysis of Ak1 and FLAG
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Frozen 9 dpf larvae were homogenized and sonicated in 4× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Samples were quantified using Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Twenty-five micrograms of protein per sample were separated by SDS-PAGE and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Ak1 polyclonal primary antibody (1:500) (14978-1-AP; Proteintech, Rosemont, IL, USA) was dissolved in 5% skim milk in TBST and incubated for 1 h at room temperature. After washing the membrane with TBST, the membranes were incubated with secondary rabbit antibody (1:1000) (NA934-100UL; GE Healthcare, Chicago, IL, USA) dissolved in 5% skim milk in TBST for 1 h at room temperature. The membrane was washed with TBST and incubated with ECL prime (GE Healthcare) for 5 min at room temperature and imaged on a LuminoGraph II EM (ATTO, Tokyo, Japan). After eliminating signals using hydrogen peroxide, the membrane was treated with FLAG (DDDDK) tag primary monoclonal antibody (1:1000) (M185-3S; MBL, Nagoya, Japan) followed by secondary mouse antibody (1:1000) (NA931-100UL; GE Healthcare).
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