Western Blot Analysis of Recombinant CFTR

Western blot analysis of cells expressing recombinant CFTR was performed using methods reported earlier (44 (link), 45 (link)). Briefly, cells were solubilized in sample buffer (46 (link)), sonicated, and heated at 37°C for 5 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7% gels. Proteins were transferred to nitrocellulose by electroblotting, and detected using either anti-RGSHis₄ (Qiagen) or R1104 anti-CFTR mouse monoclonal antibody recognizing an epitope comprising amino acids 722-734 in the CFTR regulatory (R) region (47 (link)). Horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech) was used as secondary antibody with Immobilon Western Chemiluminescent substrate (Millipore). The chemiluminescent signal was captured and analyzed on an ImageQuant LAS 4000 Mini luminescent image analyzer (GE Healthcare Life Sciences).

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Hildebrandt E., Mulky A., Ding H., Dai Q., Aleksandrov A.A., Bajrami B., Diego P.A., Wu X., Ray M., Naren A.P., Riordan J.R., Yao X., DeLucas L.J., Urbatsch I.L, & Kappes J.C. (2015). A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface. Molecular biotechnology, 57(5), 391-405.

Publication 2015

Horseradish peroxidase-conjugated goat anti-mouse IgG Immobilon Western Chemiluminescent substrate ImageQuant LAS 4000 Mini luminescent image analyzer

Amino acids Anti antibody Anti igg Antibody Buffer Cells Cftr Cftr mouse Epitope Gels Goat Horseradish peroxidase Immobilon Luminescent Monoclonal antibody Mouse Nitrocellulose Proteins Sds page Western blot analysis

Corresponding Organization : University of Alabama at Birmingham

Other organizations : Texas Tech University, Texas Tech University Health Sciences Center, University of North Carolina at Chapel Hill, University of Connecticut, Cincinnati Children's Hospital Medical Center

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Protocol cited in 3 other protocols

Variable analysis

independent variables

Expression of recombinant CFTR protein

dependent variables

CFTR protein expression measured by Western blot analysis

control variables

Cell type used to express recombinant CFTR
Solubilization and sample preparation methods (e.g., sample buffer, sonication, heating)
SDS-PAGE conditions (7% gels)
Protein transfer to nitrocellulose membrane
Primary antibodies used for detection (anti-RGSHis4 or R1104 anti-CFTR)
Secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG)
Chemiluminescent detection system (Immobilon Western Chemiluminescent substrate)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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