Cells were lysed with lysis buffer of M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific cat. no. 78501) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (100×) (Thermo Fisher Scientific cat. no. 78442). SDS/PAGE separated samples, and separated proteins were transferred to nitro-cellulose membranes and identified by immunoblotting. Primary antibodies were obtained from commercial sources and were diluted to the ratio of 1:500 or 1:1000 according to manufacturer’s instruction. Blots were developed with Supersignal Pico or Femto substrate (Pierce). Densitometric analysis of the bands was performed with the ImageQuant program (Bio-Rad).
Halt protease and phosphatase inhibitor single use cocktail 100
Manufactured by Thermo Fisher Scientific
Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (100×) is a concentrated solution designed to inhibit a broad range of proteases and phosphatases commonly found in biological samples. It is intended for use in the preparation of cell and tissue lysates to prevent degradation of proteins during sample processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Imagequant program, by Bio-Rad (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Bolt lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Ecl select reagent, by GE Healthcare (1 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Startingblock tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin hrp conjugated antibody, by Cell Signaling Technology (1 mentions)
Anti runx2 antibody, by Abcam (1 mentions)
Imagequant las 4000 system, by Cytiva (1 mentions)
Normal rabbit igg, by Merck Group (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
4 protocols using halt protease and phosphatase inhibitor single use cocktail 100
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of RUNX2 in Osteoblasts
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Proteins were extracted from lysates of MC3T3-E1 cells cultured in osteoblast differentiation medium for 12 days via the same method described above using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) with Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (100×) (Thermo Fisher Scientific). The protein solution was centrifuged, and the supernatant was mixed with 4× Bolt LDS Sample Buffer (Thermo Fisher Scientific), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on a Bolt 4–12% Bis-Tris Plus gel (Thermo Fisher Scientific), and transferred to a polyvinylidene difluoride Membrane Filter Paper Sandwich (Thermo Fisher Scientific). The membranes were probed using an anti-RUNX2 antibody (Abcam, Cambridge, UK) and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) or an anti-β-actin HRP-conjugated antibody (Cell Signaling Technology), followed by incubation with StartingBlock (TBS) Blocking Buffer (Thermo Fisher Scientific). Subsequently, the membranes were incubated with ECL Select reagent (GE Healthcare, Little Chalfont, UK) and analyzed using an Image Quant LAS 4000 system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
3
Protein Extraction and Western Blotting
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Cells were lysed with lysis buffer of M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific cat. no. 78501) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (100×) (Thermo Fisher Scientific cat. no. 78442). SDS/PAGE separated samples, and separated proteins were transferred to nitro-cellulose membranes and identified by immunoblotting. Primary antibodies were obtained from commercial sources and were diluted to the ratio of 1:500 or 1:1000 according to manufacturer’s instruction. Blots were developed with Supersignal Pico or Femto substrate (Pierce). Densitometric analysis of the bands was performed with the ImageQuant program (Bio-Rad).
4
RIP-seq of INTS11 in UV-crosslinked MCF-7 cells
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RIP was performed on freshly isolated nuclei from MCF-7 cells (2.5 107 cells per sample) after UV cross-linking with UV254nm (0.4 J/cm2). Nuclei were lysed with polysome buffer [20 mM tris-HCl (pH 8.0), 200 mM NaCl, 2.5 mM MgCl2, and 1% Triton X-100 in DEPC water] supplemented with a cocktail of protease inhibitors [Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (100×), Thermo Fisher Scientific], 1 mM dithiothreitol, and SUPERase• In RNase Inhibitor (60 U/ml; Life Technologies) and precleared with protein A beads for 1 hour at 4°C. RIP was performed overnight at 4°C on a rotating wheel using 5 μg of the specific antibody INTS11 (Sigma-Aldrich, A107128) or normal rabbit IgG (Millipore, 12-370) used as control. On the following day, 50 μl of protein A Dynabeads (Invitrogen) were coupled to the antibody for 3 hours at 4°C. The beads were rinsed five times with polysome buffer and split in two to either elute proteins or RNA (see RAP protocol for elution steps).
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