Western blotting was performed on PKD2L1–GFP plasmid after PURExpress protein expression in the presence of SUVs. SUV and protein mixture were separated on SDS-PAGE gel, Novex Tris-Glycine mini protein gels, 4–20%, 1.0 mm, WedgeWell format, (ThermoFisher, Waltham, Ma USA). The SDS-PAGE was run with 10 μl Spectra Multicolor Broad Range Protein Ladder (ThermoFisher Scientific, Cat. No. 26634). The gel was then transferred to Amersham Hybond P 0.45 PVDF blotting membrane (Cytiva, Cat. No. 10600029) and PKD2L1-GFP was detected with an anti eGFP monoclonal antibody (F56–6A1.2.3) (Invitrogen, Cat. No. MA1–952) diluted 1:1000 in TBS with 0.1% (v/v) Tween-20 and 5% (5/v) milk overnight at 4°C. The goat anti-mouse AF555 secondary (Invitrogen, Cat. No. A32727) diluted 1:5000 in TBS with 0.1% Tween-20 and 5% milk was incubated for 1 hour at room temperature.
Amersham hybond p 0.45 pvdf blotting membrane
Manufactured by Cytiva
Amersham Hybond P 0.45 PVDF blotting membrane is a polyvinylidene fluoride (PVDF) membrane designed for protein transfer and blotting applications. It has a pore size of 0.45 microns and is suitable for the immobilization and detection of proteins on western blots.
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2 protocols using amersham hybond p 0.45 pvdf blotting membrane
1
Western Blotting of PKD2L1-GFP Fusion Protein
2
Quantitative Analysis of Protein-Protein Interactions
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The protein samples were electrophoresed in the 6%–15% gradient polyacrylamide gels and blotted onto the PVDF membrane (Amersham Hybond P 0.45 PVDF blotting membrane, Cytiva). The proteins of interest were probed using the primary antibodies, that is, mouse anti‐Myc tag (Cell Signaling Technology), rabbit anti‐DDDDK (FLAG) tag (Genetex), rabbit anti‐histone H3 (R&D), rabbit anti‐CRABP2 (Proteintech), and peroxidase‐conjugated anti‐rabbit or anti‐mouse secondary antibodies. The proteins were then detected based on chemiluminescence (ECL Prime Western Blotting System, Cytiva) using Fusion FX7 imager (Vilber Lourmat). Regarding the densitometric analysis, immunoblot band intensities were quantified using the ImageJ software (Schneider et al., 2012 (link)). For the analysis of NR2F1 interaction with dimeric partners (NR2F1, NR2F2, and RXR) by conventional IP and GCE‐enabled photocrosslinking IP, the band intensity of the co‐IP Myc‐tagged partner was divided by the intensity of each FLAG‐NR2F1 variant (WT or mutants) (R1 = Myc‐partner/FLAG‐NR2F1). A final normalized intensity value (Rfin) presented in the plot was obtained by normalizing R1 of from each NR2F1 variant (R1var) against R1 from wild‐type NR2F1 (R1wt) (Rfin = R1var/R1wt, Rfin = 1 for NR2F1 WT).
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