Western blotting was performed as described previously [56 (link)]. Briefly, cells and exosomes were lysed by RIPA buffer containing PMSF (Qiagen) and quantified with BCA protein assay reagent (Thermo Fisher Scientific). Approximately 30 μg of total protein per lane were loaded. After electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (Millipore). All membranes were then blocked with 5% skim milk in TBST at room temperature for 1 h and incubated with the following primary antibodies: anti-CD9 (Abcam), anti-CD63 (Applied Biological Materials), anti-HSP70 (Cell Signaling) and anti-DLL4 (Millipore), at 4°C overnight. After washing three times with TBST, the membranes were probed with appropriate HRP-conjugated secondary antibodies (Cell Signaling) at room temperature for 1 h and visualized using ECL Plus kit (GE Healthcare).
Phenyl methyl sulfonyl fluoride (pmsf)
Manufactured by Qiagen
Sourced in United States
PMSF is a laboratory reagent used as a protease inhibitor. It functions by irreversibly inhibiting serine proteases, which are enzymes that play a crucial role in biological processes. PMSF is commonly used in biochemical and molecular biology applications to preserve the integrity of protein samples by preventing proteolytic degradation.
Automatically generated - may contain errors
Lab products found in correlation
Resource q anion exchange column, by GE Healthcare (2 mentions)
Streptactin superflow cartridge, by Qiagen (2 mentions)
Benzonase, by Qiagen (2 mentions)
Complete edta free, by Roche (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl plus kit, by GE Healthcare (1 mentions)
Anti hsp70, by Cell Signaling Technology (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti cd63, by Applied Biological Materials (1 mentions)
Anti cd9, by Abcam (1 mentions)
Protease inhibitor cocktail, by Qiagen (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Tissuelyzer 2, by Qiagen (1 mentions)
4 protocols using phenyl methyl sulfonyl fluoride (pmsf)
1
Western Blot Analysis of Exosomes
2
Affinity-Based Protein Complex Purification
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Purification of the protein complexes were performed at 4 °C. Cells were resuspended in lysis buffer [50 mM Tris/HCl (pH 8.3), 250 mM NaCl, 1 mM EDTA, 1 mM DTT, Protease inhibitor cocktail tablets (PIC) (Complete EDTA-free; Roche Diagnostics GmbH), 0.1 mM PMSF, 5 units/ml benzonase (Novagen)], subsequently sonicated and centrifuged for 1 h at 48,000 g. The soluble fraction was slowly (1 ml/min flow rate) applied to a 5 ml StrepTactin Superflow Cartridge (Qiagen) and washed with wash buffer (lysis buffer without PIC, PMSF and benzonase) until stable UV absorption could be observed. Peak fractions were incubated with TEV protease at 4 °C overnight and wash buffer without NaCl (buffer A) was used for a two-fold dilution before loading onto a ResourceQ anion-exchange column (GE Healthcare) the next day. After a washing step the complexes were eluted using a gradient with buffer B [20 mM Tris/HCl (pH 8.0), 1 M NaCl, 1 mM DTT]. A final size exclusion step on a Superose 6 Increase 10/300 GL column with 20 mM Hepes-NaOH (pH 7.8), 200 mM NaCl and 1 mM DTT was performed.
3
Affinity-Based Protein Complex Purification
4
Whole Cell Extract Preparation for Western Blot
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Preparation of whole cell extracts for Western blots was conducted according to the method we previously described (Budziszewska et al., 2010 (link)). Briefly, tissues in ice-cold RIPA buffer, containing 100 μl of Phosphatase Inhibitor Cocktail 1 and 2, 100 μl of Protease Inhibitor Cocktail, 50 μl of PMSF and OVS up to a total volume of 5 ml (all reagents, Sigma Aldrich, USA), were homogenized using a TissueLyzer II (Qiagen, USA). Samples were shaken in an ice bath for 30 min, centrifuged at 14,000 rpm for 20 min at 4°C and the supernatants were collected. Protein concentrations in the lysates were determined by the method described in Lowry et al. (1951 (link)). The cell extract concentrations were standardized by dilution with lysis buffer to the lowest protein concentration obtained. For quantitative RT-PCR, freshly isolated hippocampus and frontal cortex tissue samples were immediately placed in RNALater® solution (Applied Biosystems, USA) and stored at −80°C prior to total RNA extraction.
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