B. thetaiotaomicron strains were grown in MM containing 5 mg ml−1 maltose and were harvested at O.D.600=0.6. Cells were lysed in SDS sample buffer and 15 µg of total protein was loaded into an SDS-PAGE. SusF was detected in B. thetaiotaomicron whole cell lysates by western blot using custom rabbit polyclonal primary antibodies and horseradish peroxidase conjugated goat anti-Rabbit IgG secondary antibody (Sigma) (Cameron et al., 2012 (link)).
Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody
Manufactured by Merck Group
Horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody is a laboratory reagent used to detect the presence of rabbit primary antibodies in various immunoassays. The antibody is conjugated to the enzyme horseradish peroxidase, which catalyzes a color-producing reaction when exposed to the appropriate substrate.
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4 protocols using horseradish peroxidase conjugated goat anti rabbit igg secondary antibody
1
Detecting SusF in B. thetaiotaomicron
2
Quantifying Protein Expression by Western Blot
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Equal amount of depleted serum of each groups was pooled and then separated by 11% one-dimensional SDS-PAGE and transferred to nitrocellulose membranes in a transblot electrophoresis transfer cell (Bio-Rad, Hercules, CA, USA). Western blot analyses were performed by using rabbit polyclonal antibodies against Tf (diluted 1 : 1000, Abcam) and A1AT (diluted 1 : 500, Sigma-Aldrich). The membranes were blotted with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (diluted 1 : 20000, Sigma-Aldrich) for 1 h and then detected with enhanced chemiluminescence reagents for 1 min. Band intensities were quantified using “GelQuant.NET software provided by http://www.biochemlabsolutions.com/ .”
3
Quantification of TMEM16A Protein in Nasal Epithelia
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Nasal epithelial cells from confluent transwells (6.5 mm diameter, 0.4 μm pore size) were each lysed in 150‐μl modified radioimmunoprecipitation assay buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4, 0.2% (v/v) SDS, and 0.1% (v/v) Triton X‐100) containing a protease inhibitor cocktail (Roche) for 10 min, and the soluble fractions (20 μl) were analyzed by SDS–PAGE on 6% gels as described above and previously (Molinski et al, 2014 ; Pasyk et al, 2015 ). After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated in 5% (w/v) milk, and TMEM16A bands were detected using the human anti‐TMEM16A rabbit mAb SP31 (1:100, Abcam), using horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (1:2,500), and by exposure to film for 0.5 to 5 min as required. CNX was used as a loading control and detected using a CNX‐specific rabbit Ab (1:5,000, Sigma‐Aldrich), using horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (1: 5,000) and by exposure to film for 0.5–5 min as required. Relative levels of CFTR proteins were quantitated by densitometry of immunoblots using ImageJ 1.42 Q software (National Institutes of Health), and reported values are normalized to CNX expression levels.
4
CFTR Protein Quantification in Nasal Cultures
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Nasal cultures were grown at 37°C for 48 h in the absence or presence of small molecules (3 μM VX‐809, 1 μM VX‐661, or 4 mM 4‐phenylbutyrate, 4‐PBA) as required. Cells were then lysed in modified radioimmunoprecipitation assay buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4, 0.2% (v/v) SDS, and 0.1% (v/v) Triton X‐100) containing a protease inhibitor cocktail (Roche) for 10 min, and the soluble fractions were analyzed by SDS–PAGE on 6% gels as described above and previously (Molinski et al, 2014 ; Pasyk et al, 2015 ). After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated in 5% (w/v) milk, and CFTR bands were detected using the human CFTR‐MSD1‐specific (amino acids 25–36) murine mAb MM13‐4 (1:200, Abcam, Cambridge, UK), using horseradish peroxidase‐conjugated goat anti‐mouse IgG secondary antibody (1:2,500), and by exposure to film for 0.5 to 30 min as required. CNX was used as a loading control and detected using a CNX‐specific rabbit Ab (1:5,000, Sigma‐Aldrich), using horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (1:5,000), and by exposure to film for 0.5 to 5 min as required. Relative levels of CFTR proteins were quantitated by densitometry of immunoblots using ImageJ 1.42 Q software (National Institutes of Health), and reported values are normalized to CNX expression levels.
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