Cells were lysed at 48 hpt or 16 hpi in RIPA buffer (Abcam, Cambridge, UK) with cOmplete mini EDTA free protease inhibitor cocktail (Roche, Basel, Switzerland ) and PhosStop (Roche). Cell lysates were separated on a 10% polyacrylamide gel, followed by blotting on PVDF membrane (GE healthcare, Piscataway, NJ, USA). Regular blots were blocked in 5% nonfat milk diluted in 0.1% Tween-20 in PBS for 1 h at room temperature. When using phosphorylated protein-specific antibodies, 5% bovine serum albumin (MP Biomedicals, Santa Ana, USA) diluted in 0.1% Tween-20 in PBS was used for blocking. Primary antibodies were incubated overnight at 4 °C (Table 3 ). Following 3 consecutive 5 min washes in PBS-T, the membranes were incubated with the secondary antibody for 1h at room temperature. Following 3 more 5 min washes, the blots were detected using Pierce enhanced chemiluminescence (ECL) substrate (ThermoFisher, Waltham, MA, USA), ECL Plus substrate (GE Healthcare, Piscataway, NJ, USA), or SuperSignal West Femto maximum sensitivity substrate (ThermoFisher, Waltham, MA, USA) on a ChemiDoc MP imaging device (Bio-Rad, Hercules, CA, USA).
Chemidoc mp imaging device
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc MP imaging device is a versatile laboratory equipment designed for imaging and analysis of various types of samples, including Western blots, gels, and chemiluminescent and fluorescent samples. The device utilizes a high-resolution camera and advanced optics to capture high-quality images, and its automated functionalities enable efficient data collection and processing.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus substrate, by GE Healthcare (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Pierce enhanced chemiluminescence ecl substrate, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Abcam (2 mentions)
Phosstop, by Roche (2 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (2 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Rabbit anti human gapdh, by Cell Signaling Technology (1 mentions)
Rabbit anti mouse cd11b, by Abcam (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Nonidet p 40, by Roche (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Hrp conjugated goat anti mouse igg secondary antibody, by Agilent Technologies (1 mentions)
4 protocols using chemidoc mp imaging device
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of NLRP6 Protein
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The tissue was rinsed in saline and processed in RIPA or urea buffers. Protein samples were separated on SDS–PAGE gels under reducing conditions and transferred to nitrocellulose membranes, blocked for 1 h in 5% BSA or milk proteins diluted in PBS or TBS containing 0.5% or 0.1% Tween 20, respectively. Following blocking, membranes were incubated at 4°C overnight with the following antibodies: rabbit anti-mouse Nlrp6 (E-20; Santa Cruz), mouse anti-human NLRP6 (Clint-1; Adipogen), rabbit anti-human GAPDH (Cell Signaling), mouse anti-human tubulin (Sigma-Aldrich), mouse anti-human NLRP6 (R&D Systems), rabbit anti-GFP (Thermo Fisher Scientific), rabbit anti-mouse CD11b (Abcam), and mouse anti-mouse SMA (Clone 1A4; Sigma-Aldrich). Membranes were incubated for 1 h at room temperature with the secondary antibody at 1:5,000 in blocking buffer and visualized using ECL Western blotting detection reagents (Amersham, GE Healthcare). Images were captured using a ChemiDoc MP imaging device (Bio-Rad Laboratories).
3
Western Blot Analysis of YTH and CNOT Proteins
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Cells were lysed at 16 hpi in RIPA buffer (Abcam) with cOmplete mini EDTA free protease inhibitor cocktail (Roche) and PhosStop (Roche). Cell lysates were separated on a 10% polyacrylamide gel, followed by blotting on PVDF membrane (Amersham). Blots were blocked in 5% nonfat milk diluted in 0.1% Tween 20 in PBS for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C. Following 3 consecutive 5 min washes in 0.1% Tween 20 in PBS, the membranes were incubated with the secondary antibody for 1 h at room temperature. Following 3 more 5 min washes, the blots were imaged using Pierce enhanced chemiluminescence (ECL) substrate (Thermo Scientific), ECL Plus substrate (GE Healthcare), or SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific) on a ChemiDoc MP imaging device (Bio-Rad). Western blot assays were performed using primary antibodies against alpha-tubulin (Abcam ab40742, 1/1,000), YTHDF1 (Proteintech 17479-1-AP, 1/1,000), YTHDF2 (Proteintech 24744-1-AP, 1/1,000), YTHDF3 (Proteintech 25537-1-AP, 1/1,000), YTHDC1 (Abcam ab122340, 1/250), YTHDC2 (Proteintech 27779-1-AP, 1/500), CNOT1 (Proteintech 14276-1-AP, 1/1,000), PRV US3 (Leigh Anne Olsen and Lynn Enquist Princeton University, USA,76 (link) 1/100) and PRV gE (Hans Nauwynck Ghent University, Belgium,75 (link) 1/100).
4
Western Blot Analysis of IκBα
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Cell lysates were collected in ice-cold lysis buffer consisting of TNE (Tris-NaCl EDTA) buffer, pH 7.5, and 1% Nonidet P-40 (catalog number 37129000; Roche) in the presence of the protease inhibitor cocktail (cOmplete mini EDTA free; catalog number 11836170001; Roche). The procedure for SDS-PAGE and Western blotting is extensively described in Deruelle et al. (47 (link)). Blots were blocked in 5% (wt/vol) nonfat dry milk diluted in 0.1% phosphate-buffered saline (PBS)-Tween 20 (PBS-T) for 1 h at room temperature. The primary antibodies, the mouse monoclonal anti-IκBα antibody (clone L35A5; 1:1,000 dilution; catalog number 4814; Cell Signaling Technology), and the mouse monoclonal horseradish peroxidase (HRP)-conjugated anti-α-tubulin antibody (clone DM1A; 1:2,000 dilution; catalog number ab40742; Abcam) were incubated overnight at 4°C diluted in blocking buffer. After three 10-min washing steps with PBS-T, IκBα blots were incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:2,000 dilution; catalog number P0447; Dako) for 1 h at room temperature. Protein bands were visualized via chemiluminescence, using either ECL Plus substrate (GE Healthcare) or SuperSignal West Femto maximum-sensitivity substrate (Thermo Scientific), with a ChemiDoc MP imaging device (Bio-Rad).
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