EveryBlot Blocking Buffer

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 eliminates the traditional 1-hour incubation step while maintaining the high sensitivity of ELISA assays.

Features & 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 and within seconds 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

ELISA Assays

Indirect ELISA

Sandwich 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

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. Another 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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Related Categories

• Western Blotting Membranes
• Immunodetection Reagents and Kits
• Western Blotting Products
• Protein Electrophoresis and Blotting

Supporting Documents

• EveryBlot Blocking Buffer Flyer