What is the blotting technique？

In molecular biology and genetics, blotting technique is an analysis technology used for the detection of specific bio-molecules (proteins, DNA or RNA) in samples of complex composition The principle is to first separate the proteins, DNA or RNA by electrophoresis according to the size of the molecular,then transfer them to specific carriers, such as nylon membranes, NC transfer membranes and PVDF transfer membranes, and finally perform calibration, color development and detection on the carriers. Depending on the target object of the research, it is mainly divided into Southern Blotting for DNA testing and analysis, Northern Blotting for RNA analysis, and Western blotting for protein testing and analysis. The protein blotting is further classified into Western blotting and Eastern Blotting  based on the means of separation is unidirectional electrophoresis separation or bidirectional electrophoresis separation techniques  
Western Blotting:The fundamental principle of Western Blotting involves the specific interaction between antigens and antibodies. Proteins are separated via gel electrophoresis according to different molecular and then transferred onto a membrane. The proteins bind to the membrane through hydrophobic interactions or electrostatic forces. Before the primary antibody，we should block the non-specific binding point of the membrane with blocking agents. The target protein is then position using a primary antibody that specifically recognizes the target protein. A secondary antibody that specifically recognizes the primary antibody (which is usually an enzyme protein convenient to color development )is used to position the primary antibody then the target protein is also been position. Finally, the target protein is position and analyzed through color development including chemiluminescence, fluorescence, or staining methods. The overall experimental procedures are as follow: electrophoresis separation, transfer protein, blocking, primary antibody incubation, secondary antibody incubation, color development, detection and analysis.The detail flow diagram shows in figure1.

Figure 1：Diagram of Western Blotting

Southern Blotting：The principle of Southern Blotting is based on the base-pair complementarity of DNA double-strands. If complementary sequences exist between the DNA single-strands of two organisms, DNA double-stranded can form. If one DNA single-strands is labeled with a probe, the another DNA single-strands which is the target DNA can also been labeled to detection and analysis. Similar to Western Blotting,Southern DNA fragments are first separated using gel electrophoresis according to their sizes.What is different is that the DNA should undergo the denaturation, from DNA double-strands into DNA single-strands，then transferred onto a solid support. Non-specific binding sites on the membrane are blocked using a pre-hybridization solution. The single-stranded DNA labeled with probes is hybridized with the target DNA on the membrane，then excess hybridization solution is washed off and the hybridized DNA is detected and analyzed through colorimetric or other visualization methods. The entire experimental procedures includes DNA preparation, electrophoresis separation, membrane transfer, pre-hybridization, hybridization, washing, signal development, and imaging analysis.The detail flow diagram shows in figure2.

Figure 2：Diagram of Southern Blotting

Northern Blotting：The principle and method of Northern Blotting are basically the same as those of Southern Blotting，so there is no need to describe it detailed.

What is the blotting membrane？

After knowledge the principle and diagram of the Blotting experiment, we clearly understand that proteins or DNA, RNA are transferred to the solid-phase support along a direction perpendicular to the gel plane during the transfer process. Therefore, their positions on the support are exactly the same as those on the gel, thus it is called blotting,then the solid-phase support is the Blotting membrane. In blotting experiments, there are mainly three types of membranes commonly used as the carriers of proteins, DNA and RNA， PVDF transfer membrane, NC transfer membrane and positively charged nylon transfer membrane. Taking the Western Blotting experiment as a example，the detailed transfer process is follow:

Figure 3：Process of transfer in Blotting

Moreover, in the diagram of Western Blotting, we can clearly see that all experiments are carried out on the transfer membrane after the target has been transfer to the blotting membrane, including the blocking、primary antibody incubation、 secondary antibody incubation,、color development、film development、 detection and analysis.So the performance and compatibility of the blotting membrane will greatly affect the blotting experimental results. Therefore, the selection of the blotting membrane is very important.

How to choose the blotting membrane in the blotting experiment？

In order to achieve better experimental results, when choosing the blotting membrane in the blotting experiment, we need to consider the following precautions:

1. The solvent resistance and mechanical strength of the blotting membrane：The target substances on the blotting membrane need to be repeatedly washed and soaked for a long time with chemical reagents. Moreover, sometimes there are multiple target substances on the blotting membrane, which need to be analyzed and tested one by one. Hybridization labeling and elution need to be repeated. Therefore, when choosing a blotting membrane, we should pay attention the solvent resistance and repeatability. For example, NC membranes have relatively poor mechanical strength and inferior chemical stability compared to PVDF. Thus, in protein sequencing, the only option is the PVDF transfer membrane.

2. Membrane wettability: In the blotting experiment, the blotting membrane needs to be wetted with organic solvents or buffer solutions before use.

3. Membrane pore size: In blotting experiments, different pore sizes of target substances require blotting membrane of different specification. For example, in Western blotting, a 0.45 μ m blotting membrane is generally suitable for protein with a molecular weight greater than 20kDa, while a 0.22 μ m blotting membrane is used for protein with a molecular weight less than 20kDa. In southern blotting, for DNA fragments above 300bp, a blotting membrane of 0.45um is used, while for those below 300bp, a blotting membrane of 0.22um is used.

4. The binding ability of blotting membranes: The reason why we can detection and analysis the target substances on the blotting membrane is that the target substances can be firmly bind to the blotting membrane through hydrogen bonds, hydrophobic bonds and adsorption. Therefore, the ability of membrane to bind proteins DNA and RNA is great significance. For instance, PVDF and NC blotting membrane, due to their hydrophobic and hydrogen bonds on the membrane surface, are suitable for specific binding with proteins. In contrast, positively charged Nylon membranes with high positively charge are ideal for the adsorption binding of DNA and RNA. Moreover, when it comes to binding proteins, the protein binding ability of PVDF membranes is stronger than that of NC membranes, making them particularly suitable for protein sequencing.

5. Blocking effect: Different materials of blotting membrane have different blocking effects in the blotting experiment. For instance, among the three commonly used blotting membrane, the NC membrane has the best blocking effect and the lowest background. The positively charged nylon membrane, due to the positively charge, has a lower blocking effect than the NC membrane and the PVDF membrane, with a higher background.

6. Color development: In blotting experiment, color development mainly includes three methods: color development detection, chemiluminescence detection, and fluorescence detection. Different materials of membranes can withstand different color development methods and sensitivities. For example, nylon positively charged membranes cannot undergo color development reactions with anionic dyes.

The following table1 shows the advantages and disadvantages of the three commonly blotting membrane which can be a reference for choosing the blotting membrane in the blotting experiment.

Table 1 ：Advantages and disadvantages of the three blotting membrane

The blotting membrane of Membrane Solutions

At president，the blotting membranes of Membrane Solutions are mainly PVDF transfer membrane and the Ultra TransTM Nitrocellulose transfer membrane ，having high binding capacity for bio-molecules, providing excellent sensitivity and low background,which are tailor-made for speci?c applications, detection and blotting technologies having excellent performance.The pore sizes we can provide include 0.22um and 0.45um and we can also provide transfer membranes of di?erent shapes and dimensions.

If you want to knowledge more about the two kind of blotting membranes of Membrane Solutions by clicking the following link.

MS PVDF Transfer Membrane – Membrane Solutions

MS® NC Transfer Membrane – Membrane Solutions