20µl of protein extracts were resolved on 4%–20% gradient SDS-PAGE gels (Bio-Rad) and proteins were transferred for 1hr on 100V in transfer buffer (48mM Tris, 39mM Glycine, 20% methanol) to Immobilon-P 0.45µm PVDF membrane (Millipore) using Mini Protean Tetra transfer unit (Bio-Rad)). To detect CBX7 protein expression, Western blotting was performed with mouse monoclonal CBX7 Antibody (G-3) (Santa Cruz Biotechnologies, sc-376274) as primary antibody and goat-anti-mouse-HRP (Promega) as a secondary antibody. For quantitative Western blotting of DCAF12l1 protein, anti-WDR40B (Dcaf12l1) rabbit polyclonal antibody (Biorbit, orb155395) was used as a primary antibody along with anti-Ctcf rabbit polyclonal antibody (Cell Signaling Technologies, #2899) as a loading control. Goat-anti-rabbit- HRP (Promega) was employed as a secondary antibody. Protein bands were developed using Western Lightening Plus -ECL Kit (Perkin-Elmer) and the signal intensity was analyzed using Chemidoc MP Imaging System (Bio-Rad) and ImageLab Ver. 5.2.1 software (Bio-Rad). Exposures were captured on different times using ChemiDoc cumulative signal option to avoid signal saturation. Standard curves were prepared using increasing amounts of cell extract (Fig. 6 G ), to confirm a signal intensity staying in a dynamic linear range.
Immobilon p 0.45 m pvdf membrane
Manufactured by Merck Group
Sourced in Germany
Immobilon-P 0.45µm PVDF membrane is a laboratory equipment product manufactured by Merck Group. It is a polyvinylidene fluoride (PVDF) membrane with a pore size of 0.45 micrometers. The membrane is designed for use in various laboratory techniques and applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse hrp, by Promega (1 mentions)
Western lightening plus ecl kit, by PerkinElmer (1 mentions)
Goat anti rabbit hrp, by Promega (1 mentions)
Gradient sds page gel, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab76021, by Abcam (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
2 protocols using immobilon p 0.45 m pvdf membrane
1
Western Blot Protocol for CBX7 and DCAF12l1 Quantification
2
Western Blot Analysis of γ-Secretase Complex
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Protein samples (1.6 μg purified γ-secretase) were resolved by SDS-PAGE on 4–12% Bis-Tris gels (Life Technologies) using MES running buffer (Formedium) for 32 min at 200V. The gel was transferred to Immobilon-P 0.45 µm PVDF membrane (Millipore, Germany) in 1X transfer buffer (Life Technologies) supplemented with 20% methanol for 1 hr at 65V. The membrane was subsequently blocked for 1 hr at room temperature in 5% bovine serum albumin/Tris-buffered saline and Tween-20 (TBST) prior to incubation with indicated primary antibodies (anti-TMP21, ab133771, 1:500, Abcam, UK; anti-VAMP-8, ab76021, 1:5000 Abcam, UK; anti-Miner1, 13318-AP, 1:500, Proteintech, UK) overnight at 4°C. The membrane was washed with TBST and incubated with horseradish peroxidase-linked goat anti-rabbit IgG (NA934VS, GE Healthcare) for 1 hr at room temperature. The membrane was washed extensively with TBST and target proteins detected on FUJI Medical X-Ray Super RX film (100 NIF 18 x 24, Fujifilm, UK) using Amersham ECL Western Blotting Detection Reagent (GE Healthcare, UK).
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