An MSD-based ELISA was established to measure exposure of 306C7B3 in plasma and CSF mouse samples, similar to the description provided in ref. 102 (link). Briefly, MSD standard plates (MSD #L15XA-1) were coated overnight with 100 ng fibrillar human α-synuclein (diluted to 30 μl per well with PBS) at 4 °C overnight. After washing to remove unbound fibrillar α-synuclein, plasma or CSF probes were incubated in 25 μl for 1 h at room temperature (diluted with PBS containing 5% Amersham ECL Blocking Agent, GE Healthcare RNP2125), followed by additional washing steps and incubation with an anti-mouse secondary antibody conjugated with the MSD Sulfo-tag for detection. A similar protocol was established to measure the control anti-FITC antibody utilizing FITC-conjugated BSA (A23015, Molecular Probes) to capture the antibody102 (link).
Amersham ecl blocking agent
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Amersham ECL Blocking Agent is a protein-based solution used to block non-specific binding in Western blotting procedures. It helps reduce background signals and improve the specificity of antibody detection.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti lyve 1, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by GE Healthcare (1 mentions)
Reversible protein stain kit for pvdf membranes, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
3 protocols using amersham ecl blocking agent
1
MSD-based ELISA for α-Synuclein Exposure
2
Western Blot Analysis of Lymphatic Markers
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Aliquots containing 20 μg of total protein from cell lysates were subjected to SDS-PAGE; protein bands were then transferred to a membrane via the Trans-Blot TurboTM Transfer System (Bio-Rad, Hercules, CA, USA). Each membrane was blocked with Amersham ECL blocking agent (GE Healthcare Life Sciences, Piscataway, NJ, USA) and then incubated with each primary antibody—1:300 of anti-LYVE-1 (Abcam, Cambridge, UK), 1:300 of anti-VEGFR3 (Cell Signaling tec., Danvers, MA, USA), 1:300 of anti-Prox1 (Cell Signaling tec.), and 1:1000 of anti-β-actin (Cell Signaling tec.)—at room temperature for 15 min. Each membrane was then washed three times and incubated anti-rabbit horse radish peroxidase-conjugated secondary antibody (GE Healthcare Life Sciences) for 15 min. Protein bands were detected via Luminal Enhancers (GE Healthcare Life Sciences).
3
Western Blot Protein Detection Protocol
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Cells were lysed in ice-cold lysis buffer (10 mM TrisHCl pH 7.4, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 2 mM Na 3 VO 4 , 1% Triton X-100 and supplemented with protease inhibitors aprotinin (20 μg/mL) and PMSF (1 mM). Protein concentrations were estimated by Bradford assay, and equal amounts of protein were separated by SDS-PAGE on 10% or 12% acrylamide gels. Separat-ed proteins were transferred onto PVDF membrane and the blots were blocked by 2% blocking buffer (Amersham ECL Blocking Agent, GE Healthcare, Little Chalfont, UK) for 2 hr at room temperature. The membranes were probed with the primary antibody in 2% blocking buffer for 24 hr at 4°C, washed in TBST (10 mM Tris HCl pH 7.5, 0.1% Tween-20, 154 mM NaCl) 3 x 5 min, and then probed with the secondary antibody for 1 hr. After incubation membranes were washed for 3 x 5 min in TBST. Proteins were visualized by adding ECL reagents and exposing to X-ray film. To evaluate protein amounts SDS-PAGE gels were stained with SimplyBlue Safe Stain (Invitrogen). To confirm transfer onto a membrane proteins were visualized using Reversible protein stain kit for PVDF membranes (Thermo Scientific) following the manufacturer's instructions.
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