pVT-opti-mdr3

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Transformation of P. pastoris strain KM71H and expression analysis were as previously described [31], [35]. Selected strains were grown in a BioFlow IV fermentor and the proteins purified as previously described [13] with the following modifications: 10 mM DTT was included during cell breakage in a glass bead beater to fully reduce the proteins, and all buffers for membrane preparation and chromatography were supplemented with 1 mM β-mercaptoethanol and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) to keep proteins reduced.

Protocol tips

Transformation of P. pastoris strain KM71H and expression analysis were as previously described [31], [35]. Selected strains were grown in a BioFlow IV fermentor and the proteins purified as previously described [13] with the following modifications: 10 mM DTT was included during cell breakage in a glass bead beater to fully reduce the proteins, and all buffers for membrane preparation and chromatography were supplemented with 1 mM β-mercaptoethanol and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) to keep proteins reduced.

Publication protocol

Transformation of P. pastoris strain KM71H and expression analysis were as previously described [31], [35]. Selected strains were grown in a BioFlow IV fermentor and the proteins purified as previously described [13] with the following modifications: 10 mM DTT was included during cell breakage in a glass bead beater to fully reduce the proteins, and all buffers for membrane preparation and chromatography were supplemented with 1 mM β-mercaptoethanol and 0.1 mM tris(2-carboxyethyl)phosphine (TCEP) to keep proteins reduced. Proteins were concentrated to approximately 1 mg/ml using YM-100 Ultrafilters (Millipore). The concentrated protein was aliquoted and stored at −80°C. For gel filtration chromatography, protein was concentrated to 4 mg/ml and 0.5 ml chromatographed on Superose 6B (10×300 mm, GE Healthcare) in 20 mM Hepes-NaOH pH 7.4, 10% glycerol, 50 mM NaCl, 1 mM DTT and 0.2% n-Dodecyl-β-D-maltopyranoside (DDM) using an Äkta Purifier chromatography system (GE Healthcare). Pgp concentrations were routinely determined by UV spectroscopy at 280 nm using a calculated extinction coefficient of 1.28 per mg/ml. Serial dilutions of WT- and Opti-Pgp preparations were further assayed side-by-side with the colorimetric BCA protein assay (Pierce) using BSA with appropriate buffer controls as a standard; the two assays gave essentially the same results. Finally, increasing concentrations of different protein preparations were resolved side-by-side on Coomassie-stained SDS-gels, individual lanes were scanned and the amount of protein in the Pgp and other protein bands quantitated using ImageJ (http://rsbweb.nih.gov/ij/). The latter method permits visual inspection as well as quantitative validation of samples and allows for direct comparison of the Pgp content of the samples.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Eukaryotic cells - P. pastoris Pgp using pVT-opti-mdr3 from Ina L. Urbatsch, Department of Cell Biology and Biochemistry, an.

Manufacturer protocol

Download the product protocol from Ina L. Urbatsch, Department of Cell Biology and Biochemistry, an for pVT-opti-mdr3 below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - P. pastoris Pgp using pVT-opti-mdr3 from Ina L. Urbatsch, Department of Cell Biology and Biochemistry, an. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.