The purity of the AAV preparations were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, the samples were incubated with 6× Laemmle sample buffer (Bio-Rad) with 10% β-mercaptoethanol and boiled for 5 min at 100°C. The denatured proteins were applied to a 10% polyacrylamide gel and run at 120 V. The gel was washed three times with distilled water (diH2O) and stained with GelCode Blue Protein Safe stain (Invitrogen). In order to confirm and evaluate Rep and Cap expression from the new Rep hybrid plasmids, Western blot analyses were performed. For this purpose, the proteins were transferred to a nitrocellulose membrane following SDS-PAGE by electroblotting. The membrane was blocked in 6% milk in 1× phosphate-buffered saline and probed with hybridoma supernatants containing either monoclonal antibody (MAb) B1, detecting VP1, VP2, and VP3 (47 (link)), or MAb 1F, detecting Rep78, Rep68, Rep52, and Rep40 (30 (link)). After incubation with a secondary antibody with a linked horseradish peroxidase, the proteins were visualized by applying Immobilon chemiluminescent substrate (Millipore) and detection on an X-ray film.
Laemmle sample buffer
Manufactured by Bio-Rad
6× Laemmle sample buffer is a concentrated solution used for sample preparation in polyacrylamide gel electrophoresis (PAGE) experiments. It is designed to denature proteins and ensure consistent loading of samples onto the gel.
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2 protocols using laemmle sample buffer
1
AAV Preparation Purity Analysis
2
Characterizing AAV Protein Purity
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The purity of the AAV preparations were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, the samples were incubated with 6× Laemmle Sample Buffer (Bio-Rad) with 10% β-mercaptoethanol and boiled for 5 min at 100°C. The denatured proteins were applied to a 10% polyacrylamide gel and ran at 120 V. The gel was washed three times with distilled water (diH2O) and stained with GelCode Blue Protein Safe stain (Invitrogen). In order to confirm and evaluate Rep and Cap expression from the new Rep hybrid plasmids Western-blot analyses were performed. For this purpose, the proteins were transferred to a nitrocellulose membrane following SDS-PAGE by electroblotting. The membrane was blocked in 6% milk in 1xPBS and probed with hybridoma supernatants containing MAb B1, detecting VP1, VP2, and VP3, (43) or MAb 1F, detecting Rep78, Rep68, Rep52, and Rep40 (29) . Following the incubation with a secondary antibody with a linked horse radish peroxidase the proteins were visualized by applying Immobilon™ Chemiluminescent Substrate (Millipore) and detection on an X-ray film.
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