To test this hypothesis, human Apaf-1 was overexpressed in HEK293T cells, and then the cell lysates were used for a series of pull-down assays. By performing competition experiments, we found that increasing am
Proteins were eluted at a rate of 0.3 ml/minute and 0.5 ml fractions were collected. Equal amounts of fractions corresponding to peaks were separated by SDS-PAGE, and the proteins were detected by Coomassie staining. Biotin-DNA pull-down assays Streptavidin T1 Dynabeads (Life Technologies) were...
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A, ApoE3, and ApoE4 primary astrocytes were labeled with biotin, and the membrane proteins were purified with Avidin agarose beads. Phosphorylated and total cPLA2 levels were detected by western blot. Beta-actin was used as the loading control for cytosolic fraction, and Na,K ATPase, was the...
DNP-Biotin conjugation and purification DNP-Biotin was produced via copper free click chemistry. Briefly, Biotin-PEG2-Amine (10 mM final concentration, TCI Chemicals, Tokyo, Japan) and N-Succinimidyl N-(2,4-dinitrophenyl)-6-aminocaproate (DNP-NHS-ester; 12.5 mM final concentration, Sigma...
For volatile organic compounds (VOCs) to be released from the plant cell into the atmosphere, they have to cross the plasma membrane, the cell wall, and the cuticle. However, how these hydrophobic compounds cross the hydrophilic cell wall is largely unkn
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Selected sections from normal and fibrotic kidneys were also paraffin embedded after fixation in formaldehyde using a standard avidin-biotin complex (ABC) method, as described previously27. For morphometric evaluation, all tissue specimens were fixed in 4% buffered formalin solution (pH 7.4) and embed...
as all we as the input of total testicular lysates used in the assays.gSchematics showing the immunoprecipitation-based identification of proteins interreacting with the N-terminus of SPEM1. The biotin–streptavidin system was utilized to bind the N-terminus of SPEM1 to magnetic beads. The major...
Run-on reactions were performed at 30 °C in 2X nuclear run-on buffer (10 mM Tris (pH 8), 10 mM MgCl2, 1 mM DTT, 300 mM KCl, 20uM/ea biotin-11-NTPs (Perkin Elmer), 0.8 U/uL SUPERase-In (Thermo), 1% sarkosyl). RNA was then isolated using the Total RNA ...