Common blot-to-blot variations are accurately assessed by an internal loading control.All quantified targets' signal intensities fall within the linear range of the imaging system.The amount of protein loaded is proportional to the signal intensity.Low femtogram to high-attogram (e.g.A quantitative western blot accurately measures changes in protein expression by making relative comparisons between different treatments.Ī properly designed quantitative western blotting experiment ensures that: Sigma Chemiluminescent Peroxidase Substrate Cat# CPSOC-100ML) Bio-rad Clarity Western ECL substrate Cat#1705061) Recommended substrate for western blot detection: To detect a signal from fluorochrome-conjugated antibodies, use fluorescence imaging. To detect signals, incubate blot with chemiluminescence substrate and image.Wash the membrane 3X for 5 minutes each with Wash Buffer.Incubate membrane with secondary antibodies at room temperature for 1 hour. Wash membrane 3X for 5 minutes each with Wash Buffer.Incubate the membrane with primary antibodies at room temperature for 1 hour.Dilute primary antibodies in the blocking buffer.Recommended western blot primary and secondary antibody antibodies combinations:įinal Primary Antibody Concentration (µg/mL) Wash the membrane 2X for 5 minutes each with Wash Buffer.E.g 5 minutes for EveryBlot Blocking Buffer).
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |