SDS-rich whole cell lysates were prepared using Laemmli buffer (2% SDS, 10% glycerol) to homogenize cell layers of ALI cultures. To detect CFTR, lysates were incubated for 30 min at 37 °C prior to loading on a denaturing 6% polyacrylamide gel at 4 °C for 18 h until the 72 kDa size marker band approached end of the acrylamide matrix. Transfer to a nitrocellulose (Hybond C, GE Healthcare, Chicago, IL, USA) membrane was done immersed in an ice bath at 0 °C for 18 h. StartingBlock Block Buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used for blocking for 2 h at room temperature. CFTR detection was done using four CFTR monoclonal antibodies provided by T. Jensen, Chapel Hill via the antibody distribution program hosted by the CF foundation (see Table 2 ) in a mixture of 1:1:1:1. Depending on signal intensity, two different HRP substrate solutions (SuperSignal West Pico or SuperSignal West Femto, Thermo Fisher Scientific) were applied onto membranes. Fatty acid synthase (FASN, MW 273 kDa) was used as a loading control. Moreover, to compare non-treated controls and AAT-treated T84 cells we used paired samples from the same experiment.
Startingblock block buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
StartingBlock Block Buffer is a specialized buffer solution designed for use in various laboratory applications. It serves as a versatile reagent for blocking non-specific binding sites in immunoassays and other biochemical experiments. The buffer is formulated to effectively reduce background signals and improve the specificity of target analyte detection.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using startingblock block buffer
1
Detecting CFTR Expression in ALI Cultures
2
Quantifying Anti-ZIKV NS1 Antibody Titers
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To determine endpoint anti-NS1 antibody titers, Nunc MaxiSorp flat-bottom 96-well plates (Thermo Fisher Scientific) were coated overnight at 4°C with ZIKV NS1 protein (Sino Biological) at 1 μg/ml in PBS as described previously. The plates were washed four times with PBS +0.05% Tween 20 (PBST) and then blocked with StartingBlock Block Buffer (Thermo Fisher Scientific) for 5 min at room temperature. Serially diluted mouse serum samples were added to wells and incubated at 37°C for 1 hour, and the plates were washed four times. Bound antibodies were detected using HRP-conjugated goat anti-mouse IgG (GE Healthcare Life Sciences), and the optical density (OD) was read at 492 nm. To confirm that the IgG2a antibody isotype predominates the anti-NS1 humoral response, antibodies were captured as described above, and bound antibodies were detected using HRP-conjugated anti-mouse IgG2a (GE Healthcare Life Sciences). Endpoint titers were determined as the reciprocal of the highest serum sample dilution with an OD reading above the cutoff, set as 2SD above the mean OD of serum samples from prevaccinated or naïve mice.
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