For pull down assay, the cell lysates of pCDNA3.1-SCARB2-Myc transfected 293T cells in the radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.1% deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM Tris–HCl (pH 7.8), and 1 mM EDTA) containing a proteinase inhibitor cocktail (GE, USA), were incubated with anti-myc antibody conjugated magnetic beads (Sino Biological Inc., China) overnight at 4 °C before washing the beads three times with PBS containing 0.1% Tween 20 (v/v) (PBST). Then the EV71 particles was incubated with or without PC (100, 500 μM) at 37 °C for one hour. After that, the EV71-PC mixture was added to SCARB2-myc coupled magnetic beads and incubated at 4 °C for 2 h. After washed thrice with PBST, the VP1 protein bound to the beads was analyzed by immunoblotting with an anti-VP1 antibody, and the SCARB2-myc on the beads was detected by anti-myc antibody.
Proteinase inhibitor cocktail
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Proteinase inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteolytic enzymes, known as proteases, in biological samples. It is designed to prevent the degradation of proteins during sample preparation and analysis. The cocktail contains a mixture of chemical inhibitors that target a broad range of proteases, helping to preserve the integrity of the proteins being studied.
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Lab products found in correlation
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
Ecl prime kit, by GE Healthcare (1 mentions)
Anti p stat6 antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
2 protocols using proteinase inhibitor cocktail
1
EV71 SCARB2 Binding Inhibition Assay
2
Protein Phosphorylation Analysis by Western Blot
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Cells were washed with PBS and lysed in 1% sodium dodecyl sulfate (Sigma) with a proteinase inhibitor cocktail (GE Healthcare, Little Chalfont, England) and a phosphatase inhibitor cocktail (Roche Diagnostics, Tokyo, Japan). Samples were electrophoresed and transferred onto PVDF membranes (Millipore). After blocking with 10% bovine serum albumin (Wako, Osaka, Japan), the membrane was incubated with anti-p-STAT6 antibody (Cell Signaling Technology, Tokyo, Japan) or anti-GAPDH antibody (Cell Signaling Technology) then incubated with HRP-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA, United States). The signal was detected with an ECL prime kit (GE Healthcare, Little Chalfont, United Kingdom) and imaged using a LAS 4000 analyzer (FUJIFILM, Tokyo, Japan).
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