Protein lysates from RPE/choroid were placed in sample buffer containing dithiothreitol and boiled for 6 minutes at 95°C. Equal amounts of protein were separated using SDS polyacrylamide gel electrophoresis and transferred into a polyvinylidene difluoride (PVDF) membrane using the iBlot system (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). This membrane was blocked with a blocking buffer from Li-Cor Biosciences (Lincoln, NE, USA) for 1 hour at room temperature and incubated overnight with the designated primary antibody at 4°C. To detect MnSOD we used a primary rabbit polyclonal antibody from Abcam (Cat. no. ab13533, 1:1000 dilution; Cambridge, MA, USA) and a donkey anti-rabbit secondary antibody from Li-Cor Biosciences (Cat. no. 926-32213, 1:5000 dilution); to detect expression of the exogenously delivered Sod2-myc gene, we used a mouse anti-myc antibody from Invitrogen (Cat. no. 04-1117, 1:5000 dilution), and the secondary antibody was donkey anti-mouse from Li-Cor Biosciences (Cat. no. 926-68072, 1:5000 dilution). Rabbit anti-alpha–tubulin primary antibody from Abcam (Cat. no. ab7291, 1:10,000 dilution) and a donkey anti-rabbit secondary antibody from Li-Cor Biosciences (Cat. no. 926-32213, 1:5000 ratio) were used to detect tubulin.
Donkey anti rabbit secondary antibody
Manufactured by LI COR
Sourced in United States
The Donkey anti-rabbit secondary antibody is a reagent used in immunoassay techniques, such as Western blotting and immunohistochemistry. It is designed to detect and bind to rabbit primary antibodies, allowing for the visualization and quantification of target proteins or antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by LI COR (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Tris carboxyethyl phosphine hydrochloride, by Merck Group (1 mentions)
Mab1501, by Merck Group (1 mentions)
Sc 110, by Santa Cruz Biotechnology (1 mentions)
Amersham protran, by GE Healthcare (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Mouse anti myc antibody, by Thermo Fisher Scientific (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Antibodies against akt and phospho akt ser473, by Cell Signaling Technology (1 mentions)
Odyssey software v3, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
6 protocols using donkey anti rabbit secondary antibody
1
Western Blot Analysis of Retinal MnSOD
2
Quantitative Western Blot Analysis
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Protein was isolated from the HUVEC after incubation with CDDO-Im (200 nM) for each of the time points represented in the microarray experiment (0.5, 1, 3, 6, and 24 hours), by addition of 50 µL of lysis buffer (Life Technologies) containing 10 mM Tris (carboxyethyl) phosphine hydrochloride (Sigma-Aldrich). About 15 µL, containing approximately 5 µg of protein, from each treatment was run on E-PAGE 96-well 6% gels (Life Technologies) and then transferred to a nitrocellulose membrane (Life Technologies). After blocking in blocking buffer (LI-COR Biosciences, Lincoln, NE USA), the blots were then incubated with rabbit HMOX1, HSP105, DUSP1, DYRK3, or HSP70 primary antibodies (1:5000; Assay Designs Inc., Ann Arbor, MI, USA) for 1 hour. In addition, each blot was dual-labeled with β-actin as a loading control that was used for normalizing antibody intensity of each protein of interest. The blots were washed 3 times with 0.1% Tween 20 in phosphate-buffered saline and incubated with donkey anti-rabbit secondary antibody (LI-COR Biosciences) for 30 minutes before 3 more washes and then allowed to dry for visualization. Visualization was performed using the Odyssey Imaging System (LI-COR Biosciences) that allowed for dual labeling of 2 different proteins and was quantified by direct measurement of background-subtracted infrared florescent signals.
3
Immunoblot Analysis of HA-HC Complexes
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HA-HC complexes generated above (10 μl) and control samples were incubated or not with Strp HA’se (2 μl) in PBS to a total of 25 μl for 1 hr at 37 °C. Each sample (10 μl) was heated to 95 °C for 5 mins and loaded on to a polyacrylamide gel, transferred to a membrane, blotted with the primary anti-IαI antibody (Cleveland Clinic) followed by a LI-COR donkey anti-rabbit secondary antibody for HC detection using an Odyssey Scanner.
4
Western Blot Analysis of Egr-1 Expression
5
Western Blot Analysis of Cathepsin K in Osteoclast Lysates
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AA and SS osteoclast cell lysates were resuspended in lysis buffer for SDS-PAGE and western blotting. The Micro Bicinchoninic Acid Total Protein Assay (Pierce Chemicals) was performed, and equal amounts of cell lysate were loaded for SDS-PAGE. After electrophoresis, the proteins were transferred onto nitrocellulose membranes and incubated with rabbit anti-catK polyclonal antibody (1:500; Proteintech) overnight and with donkey anti-rabbit secondary antibody (LI-COR Biosciences). The membranes were imaged with an LI-COR Odyssey CLx (LI-COR Biosciences).
6
Western Blot Analysis of Phospho-AKT and Total AKT
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Western blot for phospho-AKT (Ser473), total AKT, and β-actin were performed on gWAT protein extracts as described [45 (link)]. Briefly, the protein concentration was measured using the DC protein assay (Bio-Rad laboratories, Veenendaal, The Netherlands), and 20 µg of a protein sample was run on a 12% acrylamide gel and blotted on a PVDF membrane (Merck Millipore, Amsterdam, The Netherlands). The membrane was incubated with primary antibodies (the antibody against β-actin was purchased from Abcam, Cambridge, UK; antibodies against AKT and phospho-AKT (Ser473) were purchased from Cell Signaling Technology, via BIOKÉ, Leiden, The Netherlands) at 4 °C overnight and then was incubated with goat anti-mouse secondary antibody for β-actin or donkey anti-rabbit secondary antibody (LI-COR, Lincoln, NE, USA) for the other antibodies, all at room temperature for 1 h. The membrane was scanned on an Odyssey scanner (LI-COR, Lincoln, NE, USA). Bands were analyzed using Odyssey software V3.0 (LI-COR, Lincoln, NE, USA).
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