Criterion any kd precast gels

Manufactured by Bio-Rad

The Criterion Any-kD precast gels are versatile polyacrylamide gels designed for protein electrophoresis. These gels feature a gradient composition that allows for the separation of a wide range of protein molecular weights in a single run.

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4 protocols using criterion any kd precast gels

SDS-PAGE Coomassie Blue Gel Imaging

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SDS-PAGE Coomassie blue gels were run using Criterion Any-kD precast gels (Bio-Rad). The gels were run for 1 h at 120 V using a PowerPac source (Bio-Rad). Next, gels were rinsed in ultrapure water twice for 5 min and stained using Bio-safe Coomassie G-250 stain (Bio-Rad) for 1 h. Gels were de-stained in ultrapure water for 30 min and imaged using a ChemiDoc MP imager (Bio-Rad). Kinetic endpoint gels were run at the end of the drug-seeded aggregation assay by combining the triplicate wells within each experiment and loading a total of 8 μg (monomer equivalent). All gels included Precision Plus Dual Color protein ladder (Bio-Rad) as a reference.

Kochen N.N., Vasandani V., Seaney D., Pandey A.K., Walters M.A., Braun A.R, & Sachs J.N. (2022). Threonine Cavities Are Targetable Motifs That Control Alpha-Synuclein Fibril Growth. ACS chemical neuroscience, 13(17), 2646-2657.

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Western Blot Protein Analysis Protocol

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For Western Blot analyses, proteins were extracted from cultured cells with a Cell Lytic solution (Sigma-Aldrich) following the manufacturer protocol and protein concentration was then measured by a BCA assay. 20 μg of proteins were separated on SDS-PAGE (Criterion any KD precast gels, BioRad) and electro-transferred onto Hybond nitrocellulose membranes. Membranes were then blocked with the Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE, USA) and incubated with Santa Cruz antibodies (Dallas, Texas, USA), p16 (sc-448) and Nrf2 (sc-365949), Enzo Life Sciences antibodies (Villeurbanne, France), either proteasome β1 (BML-PW8140) and β5 (BML-PW8895) and immunoproteasome β1i (BML-PW8205), β2i (BML-PW8150-0100) and β5i (PW8200) catalytic subunits and proteasome regulators PA28αβ (BML-PW185-0100, PW8240) at dilutions of 1/500 for Nrf2, 1/1000 for p16, β1, β2, β5, PA28αβ and β2i, 1/2000 for β1i and 1/5000 for β5i. Then, membranes were washed with PBS and incubated with species infraredconjugated secondary antibodies (Li-Cor Biosciences) at a dilution of 1/15000 and scanned with the Odyssey infrared imaging system (Li-Cor Biosciences). Signal intensities were quantified with the ImageJ software.

Desvergne A., Ugarte N., Radjei S., Gareil M., Petropoulos I, & Friguet B. (2016). Circadian modulation of proteasome activity and accumulation of oxidized protein in human embryonic kidney HEK 293 cells and primary dermal fibroblasts. Free radical biology & medicine, 94.

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Immunoblot Analysis of Borrelia burgdorferi

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B. burgdorferi B31 whole-cell lysate (WCL) was generated and fractionated by SDS-PAGE using Any-Kd Criterion Precast gels (Biorad) and standard methods (Izac et al., 2017 (link)). Proteins were visualized with Coomassie brilliant blue (CBB-R 250) or transferred to PVDF membranes (Pierce) for immunoblot analyses (Oliver Jr et al., 2016 ). Dot-blot procedures were as previously described (Oliver Jr et al., 2016 ). In brief, 125 ng of protein was spotted onto nitrocellulose and dried overnight. Equal protein loading was confirmed by staining (MemCode Reversible Protein Stain Kit; Thermo Scientific). Immunoblots and dot-blots were screened with diluted sera (1:1,000 and 1:200, respectively) and Ab binding detected using horseradish peroxidase (HRP)-conjugated anti-dog IgG Ab (1:40,000; Pierce) and Clarity Western ECL Substrate (Biorad). Images were captured using a ChemiDoc imaging system (Biorad). All blots from a given experiment were imaged simultaneously using identical parameters. The dot-blot images were cropped into a grid for visualization and comparison. All experiments were performed three times.

Hatke A.L., Green D.R., Stasiak K, & Marconi R.T. (2020). Antibody profiling of a Borrelia burgdorferi C6 antibody positive, clinically affected Rottweiler and her pups. Veterinary journal (London, England : 1997), 262, 105504.

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Western Blot and Dot-Blot Analysis

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Cell lysates (0.3 OD₆₀₀ units per lane) were separated using AnykD Criterion Precast Gels (Bio-Rad) and transferred to PVDF membranes using the Transblot Turbo System per the manufacturer’s protocol (Bio-Rad). Dot-blots were generated by spot application of 500 ng of GspD, LipL32, and FhbB (negative control) on to nitrocellulose membranes (22 µM pore size; BioRad) followed by air drying. Immunoblots and dot-blots were incubated with blocking buffer (PBS with 5% nonfat dried milk; 0.2% Tween 20; 2 h). Microscopic agglutination test (MAT) positive canine sera (1:200 dilution) were added (1 h), the blots were washed and rabbit anti-canine IgG HRP-conjugated secondary antibody (1:20000 dilution; Novus Biologicals) was added. IgG binding was detected using ECL substrate (Bio-Rad) and images were captured using a ChemiDoc Touch Imaging System (Bio-Rad). Note that the immunoblot and dot-blot images were cropped to generate the figures.

Schuler E, & Marconi R. (2021). The Leptospiral General Secretory Protein D (GspD), a secretin, elicits complement-independent bactericidal antibody against diverse Leptospira species and serovars. Vaccine: X, 7, 100089.

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