EveryBlot Blocking Buffer

EveryBlot Blocking Buffer

EveryBlot Blocking Buffer rapidly blocks western blots in 5 minutes and enhances sensitivity, regardless of the detection method. It’s also ELISA-compatible, reducing blocking time from 1 hour to zero minutes.

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EveryBlot Blocking Buffer provides 5-minute blocking and maximum sensitivity for all western blots regardless of detection method. This versatile blocking buffer is compatible with Direct and Indirect ELISA applications. EveryBlot Blocking Buffer requires no incubation time, eliminating the traditional 1-hour incubation step while maintaining the high sensitivity of ELISA assays

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
  • Compatible with Sandwich and Indirect ELISA assays
  • Rapid blocking - 5 minutes for Western Blots & 0 Minutes for ELISA assays

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.

NEW! Download the EveryBlot Blocking Buffer for ELISA Assays protocols:

ELISA Assays

Indirect ELISA

Chart for Indirect ELISA

Sandwich ELISA

Chart for Direct ELISA

Blocking Buffer Comparison. EveryBlot Blocking Buffer and standard ELISA BSA Block or Competitor ELISA blocking buffer showed comparable results with good correlation coefficients.

Fluorescent Detection

Clarity Western ECL Substrate

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

Clarity Max Western ECL Substrate

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

Low inherent fluorescence

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 ().

Packaging Options

Related Products

  • 1x Tris Buffered Saline (TBS) with 1% Casein (1610782)
  • 1x Phosphate Buffered Saline (PBS) with 1% Casein (1610783)
  • Blotting-Grade Blocker (1706404)
  • Gelatin (1706537)
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Supporting Documents

  • EveryBlot Blocking Buffer Flier



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Bottle of blocking buffer, 500 ml. Requires only 5 minutes of blocking for all western blots and eliminates incubation time in ELISA assays.


Number Description Options
EveryBlot Blocking Buffer Flier
EveryBlot Blocking Buffer Instructions Flier
EveryBlot Blocking Buffer for Indirect ELISA Protocol
EveryBlot Blocking Buffer for Direct ELISA Protocol