Takara Protein Detection Utility makes Western Brotting experiments easy!

Western blotting is a commonly used experimental method
in molecular biology.
The basic principle is to separate the protein sample by polyacrylamide gel electrophoresis, transfer it to a solid-phase support (such as cellulose film), and use the antigen-antibody reaction to detect the protein
of interest.
The general operating process of Western Blotting is as follows:

Today I will introduce some Western Blotting-related Takara protein detection utilities to help you improve the success rate of your experiments!

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Choice of blocking reagent: In this step, it is important
to block the membrane region of the unbound protein in order to prevent the protein detection antibody from binding to the membrane non-specifically.
Sometimes the use of a specific blocking agent may not achieve the desired effect, so several blocking agents can be prepared for point hybridization pre-experiments to determine the appropriate blocking agent
.

Common blocking agents include skimmed milk powder and BSA, but these blocking agents contain mammalian-derived components and may non-specific cross-reactivity
with mammalian-derived antibodies.
Takara offers two blocking agents without ingredients of mammalian origin, giving you more blocking agent options:

(1) Western BLoT Blocking Buffer (Fish Gelatin) (Code No.
T7131A), this product uses fish-derived gelatin as a blocking agent, compared with mammalian-derived milk powder and BSA, it will not produce non-specific cross-reactivity with mammalian-derived antibodies, and can clearly detect specific antigens
.
Because it is a ready-to-use reagent, it does not need to be diluted, so it can be used directly to block Western blotting membranes; It can also be used as a dilution
for primary antibody, HRP, or AP-labeled secondary antibody.

(2) Western BLoT Blocking Buffer (Protein Free) (Code No.
T7132A) is a blocking buffer composed of chemical composition, does not contain proteins, can inhibit non-specific signals well, and can be effectively used to detect phosphorylated proteins
.
This is also a ready-to-use reagent
.

Primary antibody reaction → wash→ secondary antibody reaction → wash

Stripping Buffer: Stripping Buffer refers to a reagent
that removes primary and secondary antibodies from the membrane, but does not affect the protein to be measured bound on the membrane.
Due to the different concentrations of the target protein and the antibody concentration used, the film exposure conditions are also different, so the use of this more characteristic antibody stripping reagent can effectively remove the antibodies on the membrane, help everyone repeatedly explore the experimental conditions, and save antibodies
.

Western BLoT Stripping Buffer (Code No.
T7135A), which can help you re-investigate and optimize experimental conditions
by removing the primary antibody, secondary antibody, and detection reagent from the membrane at room temperature, and then re-detecting the protein of interest and other proteins on the same membrane.

Antigen-antibody reaction promoter: It can significantly improve the detection sensitivity, even if there is only a small amount of antibody, it can effectively carry out antibody reaction and improve the detection signal strength
.

(1) Western BLoT Immuno Booster (Code No.
T7111A) is suitable for a variety of immune tests including Western Blotting and ELISA, which can improve the final detection sensitivity (from several times to tens of times).

(2) Western BLoT Immuno Booster PF (Code No.
T7115A) does not contain protein, and can be combined with Western BLoT Blocking Buffer (Protein Free) (Code No.
T7132A) for detection and analysis
under a system without foreign proteins.

Other related products: protein quantification reagents

Protein Quantitation Reagent Selection: In order to keep the loading of different samples consistent for subsequent results analysis, protein samples are often quantified prior to WB
.
Commonly used protein quantification methods are BCA method and Bradford method (Coomassie brilliant blue method), and Takara has prepared the corresponding protein quantification reagents
.

(1) TaKaRa BCA Protein Assay Kit (Code No.
T9300A) is for different kinds of protein, and the measurement value changes little
.
In the 20-2,000 μg/ml concentration range, protein concentration is linearly related to absorbance values and is less
affected by surfactants.

(2) The TaKaRa Bradford Protein Assay Kit (Code No.
T9310A) is simple and rapid, can perform protein quantification in the 1-1,000 μg/ml concentration range, and is not affected by
reducing agents.

Click on the image below to download the Western Blotting Experiment Operation Guide

For more details, please visit:
Takara Bio China