Description
Xpert Enhancer Blocking Solution is a 2-in-1 Buffer for Western Blotting; not only can membrane blocking and antibody hybridization be carried out in a single-step, saving considerable amounts of time, the buffer also enhances the signal developed with either HRP (horseradish peroxidase) or AP (alkaline phosphatase) substrates.
Xpert Enhancer Blocking Solution is a protein-free ready-to-use blocking solution that provides effective blocking with reduced overall background and non-specific signals. It allows for simultaneous blocking and antibody binding and can be used both as a 2-in-1 step procedure (blocking + primary antibody binding, followed by secondary antibody binding after washing) and as a 3-in-1 step procedure (blocking + primary antibody binding + secondary antibody binding), saving hands-on time as well as up to 2 hours of total time, whilst increasing up to 2.5-fold signal intensity.
Features
Ready-to-Use
Protein-free blocking solution
Simultaneous blocking and antibody binding Enhanced antibody signal
Time-Saving
Compatible with Nitrocellulose and PVDF membranes
Compatible with any substrate for HRP and AP
Stable at room temperature for at least 1 year
Protocols
Basic 2-step protocol
- Following Western Blot transfer, immerse the membrane (PVDF or NC) in PBST or TBST for 5 minutes.
- Mix primary antibody and Xpert Enhancer Blocking Solution. For example: in case of diluting primary antibody 10,000, add 1µl of the primary antibody to 10ml of Xpert Enhancer Blocking Solution. Thoroughly Mix.
- Remove PBST/TBST and place the membrane in the Primary Antibody-Xpert Enhancer Blocking Solution and incubate at room temperature for 1 hour with gentle agitation.
- Wash the membrane 3x with PBST/TBST
- Mix secondary antibody and Xpert Enhancer Blocking solution. For example: in case of diluting secondary antibody 10,000, add 1µl of the secondary antibody to 10ml of Xpert Enhancer Blocking Solution. Thoroughly Mix. (It is recommended to dilute secondary antibody 10,000 or more, as higher concentrations may result in higher background.
- Remove PBST/TBST and place the membrane in the Secondary Antibody-Xpert Enhancer Blocking Solution and incubate at room temperature for 1 hour with gentle agitation.
- Wash the membrane 3x with PBST/TBST
- Remove excess wash buffer and proceed with image development immediately (either with enhanced chemiluminescence (ECL) or colorimetric reagents)
1-step protocol
In order to reduce hands-on and total time even further, the whole procedure can be carried out in a single step by combining blocking and incubation with both primary and secondary antibody. Please note that in comparison with the 2-step protocol, detection sensitivity will be lower.
- Following Western Blot transfer, immerse the membrane (PVDF or NC) in PBST or TBST for 5 minutes.
- Mix primary antibody and Xpert Enhancer Blocking Solution. For example: in case of diluting primary antibody 10,000, add 1µl of the primary antibody to 10ml of Xpert Enhancer Blocking Solution. Thoroughly Mix.
- In a separated tube, mix secondary antibody and Xpert Enhancer Blocking solution. For example: in case of diluting secondary antibody 10,000, add 1µl of the secondary antibody to 10ml of Xpert Enhancer Blocking Solution. (It is recommended to dilute secondary antibody 10,000 or more, as higher concentrations may result in higher background.
- Remove PBST/TBST and place the membrane in the Primary Antibody-Xpert Enhancer Blocking Solution. Add the Secondary Antibody-Xpert Enhancer Blocking Solution and incubate at room temperature for 1 hour with gentle agitation.
- Wash the membrane 3x with PBST/TBST
- Remove excess wash buffer and proceed with image development immediately (either with enhanced chemiluminescence (ECL) or colorimetric reagents)
