A novel way to perform Western Blot

Mix OneStep Blocker, 1°Ab and 2° Ab with membrane together, that's all.

OneStep Blocker is a blocking solution for Western blot analysis.

This OneStep Blocker buffer not only provides blocking, primary and secondary antibody hybridization in one step but also enhances the signal developed with HRP (horseradish peroxidase) or AP (alkaline phosphatase) substrates. It, therefore, serves as both blocker and enhancer in Western analysis. With the 3-in-1 step procedure, OneStep Blocker is a time and labor economic solution for the time consuming and laborious Western procedure.

• 3 steps in one: Block the membrane and dilute 1^o  Ab & 2^o  Ab in one step.
• Enhance antibody signal: It shows a two- to five-fold increase in signal intensity for most protein targets, enabling much less protein to be detected with the same substrate and method.
• Enhance time-saving: It saves at least 2 hours in the antibody detection process during the Western Blot, with only one hour needed.
• Universal antibody diluent: Ready-to-use dilution buffer for most of 1^o  Ab & 2^o  Ab.
• No blocking step needed: Just immerse the membrane in the OneStep Blocker solution with your antibodies. That's all.
• Effective with any ECL: After the antibody detection process, the signal can be developed with both HRP (horseradish peroxidase) and AP (alkaline phosphatase) substrates.
• Less hands-on steps: No 3 wash steps are required, meaning no need to transfer the membrane in&out of the container.
• Compatible with PVDF & NC membrane: Regardless of the pore size, the OneStep Blocker minimizes the background from non-specific protein binding by antibodies.
• Improve protein detection: Improve the binding process of target proteins, so that specific antibodies can bind more effectively.
• Protein free: Reduces overall background and minimizes non-specific signals often seen with ECL detection.

** Required materials but not provided

＃Primary antibody.
＃Secondary antibody conjugated with HRP.
＃Wash buffer:

PBST (phosphate buffered saline with Tween-20) or TBST (Tris buffered saline with Tween-20) buffer.
ECL (Enhanced chemiluminescence) or colorimetric reagents.

＃Shaker: orbital or rocking shaker.

1. After Western blot transferring, immerse the PVDF or NC membrane in PBST buffer for 5 minutes.

2. Dilute the primary antibody and secondary antibody with proper amounts of OneStep Blocker.

• 2.1 For example, when the dilution factor for both primary and secondary antibodies is 1: 10,000, add 2μl of the primary antibody to  10ml of  the OneStep Blocker (1st tube), followed by adding 2μl of the secondary antibody to another 10ml of the OneStep Blocker (2nd tube).
• 2.2 Thoroughly mix the antibody-OneStep Blocker solution inside each tube by inverting it back and forth.
• 2.3 Pour the primary antibody-OneStep Blocker solution into the prepared container first, followed by the addition of the secondary antibody-OneStep Blocker solution into the same container.

3. Incubate the membrane immediately in the antibody-OneStep Blocker solution at room temperature for 1 - 2 hours with gentle agitation. Please note that after mixing  the primary and secondary antibodies, the membrane needs to be immediately immersed in the mixture within 10 minutes for obtaining the optimal performance.

4. Wash the membrane with PBST/TBST three times with shaking.

5. Drain excessive wash buffer and perform image development methods with ECL or colorimetric system immediately.

Can Primary Antibody & Secondary Antibody be hybridized in 1 step with great efficiency?

1. Please note that after mixing the primary and secondary antibodies, the membrane needs to be immediately immersed in the mixture within 10 minutes for obtaining the optimal performance.

2. The dilution for the secondary antibody should be at least 1:10,000 or more. A higher level of background noise will be observed as a result with a high concentration of secondary antibody.

3. Do not incubate membrane in OneStep Blocker for over 4 hours to avoid high background. Overnightincubation is especially not recommended.

4. The primary/secondary antibodies mixed in OneStep Blocker solution may be reused within 3 days. Enhancing effect may trail off along with the increasing storage time or repetitiveness. Keep the mixed solution refrigerated. For critical experiment or strong signal, fresh preparation of antibody-OneStep Blocker solution is required.

5. When the antibody concentration is too high or if the prolonged incubation takes place, it will cause high background. When excessive background occurs, please try the followings:
(a) Reduce/optimize primary and/or secondary antibody concentrations.
(b) Use dot-blot test to optimize antibody concentrations.
(c) Reduce/optimize incubation time.