EveryBlot Blocking Buffer

Overview

EveryBlot Blocking Buffer provides 5 minute blocking and maximum sensitivity for all western blots regardless of detection method.

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Description

EveryBlot Blocking Buffer provides 5 minute blocking and maximum sensitivity for all western blots regardless of detection method.

Features and Benefits

Reduces non-specific antibody binding to reduce background while maintaining excellent sensitivity
For both chemiluminescent and fluorescent detection
Contains no phosphate-based buffers. Ideal for use with phospho-specific antibodies
Complete blocking in 5 minutes

Recommended Protocol

For mini-sized blots, use at least 10 ml for blocking and antibody incubation steps.
For midi-sized blots, use at least 20 ml.

Add block to the membrane and incubate for 5 minutes with agitation
Dilute primary and secondary antibodies in full-strength block and incubate for 1 hour with agitation

For fluorescent detection on PVDF, add SDS to 0.02% in the secondary antibody solution.

Fluorescent Detection

Fluorescent multiplex detection in HEK293 cells. After electrophoresis, proteins were transferred to low fluorescence PVDF. Left, blot blocked in a fluorescent optimized blocker for 1 hour. Right, blot blocked in EveryBlot Blocking Buffer for 5 minutes. After blocking, both blots processed identically and imaged to maximize signal and minimize background for each channel independently.

Chemiluminescent Detection

Chemiluminescent detection of three targets in HEK 293 cells. After blocking, both blots processed identically and imaged and displayed using identical parameters.

Phosphoprotein Detection

Nothing to interfere with phosphoprotein detection. Detection of total (green) and phospho-specific (red) targets. Left, Jurkat cells unstimulated (–) or stimulated (+) with calyculin A. Center and right, A431 cells unstimulated (–) or stimulated (+) with EGF.

Low Background Across the Entire Spectrum

Effect of blocking agent on membrane background. Low fluorescence PVDF membranes without sample or antibodies blocked in either a fluorescence optimized blocking buffer or EveryBlot Blocking Buffer. After washing, membranes were imaged side by side on the ChemiDoc MP Imaging System. Membrane autofluorescence was measured in each fluorescent channel. Other vendor's buffer (); EveryBlot Buffer ().