What Is a Blotting Machine?
Blotting machine is a device used for transferring biological molecules (such as DNA, RNA, and proteins) from a gel after electrophoresis.
They are primarily utilized in the fields of biology and molecular biology research. Electrophoresis is an analytical technique mainly employed for the separation of proteins or DNA based on their movement properties under an applied voltage.
Firstly, in Western blotting, the separation and identification of proteins are possible. After electrophoresis, proteins are transferred onto a membrane, labeled with specific antibodies, and detected.
Uses of Blotting Machine
Blotting machines are mainly used in biological and biochemical experiments, but their applications vary depending on their types.
1. Western Blotting
Western blotting is a technique for transferring and detecting proteins separated by electrophoresis on a membrane using antibodies.
The procedure involves obtaining antibodies specific to the target protein, separating the proteins through electrophoresis, transferring the obtained bands to a membrane, and performing blocking and antibody reactions to detect the target protein.
Specifically, the protein adhered to the membrane is transferred from the gel by applying voltage, reacts with antibodies specific to the target protein (primary antibody), followed by a reaction with labeled enzymes as secondary antibodies. Finally, the target band is visualized using chemiluminescence or colorimetric methods.
2. Southern Blotting
Southern blotting is a technique used to detect specific DNA sequences, primarily employed in molecular biology experiments. DNA samples are electrophoresed on an agarose gel, transferred (blotted) onto a membrane, and then detected using labeled probes specific to the target DNA sequence.
Probes are typically labeled with radioactive isotopes or color-producing enzymes, visualizing DNA with specific base sequences. This method is used for genome mapping, individual identification, and paternity testing.
3. Northern Blotting
Northern blotting is a technique for detecting specific RNA sequences, primarily used to investigate gene expression patterns and functions. It allows the study of how DNA is transcribed into mRNA and how it functions.
The procedure involves extracting DNA and RNA from cells, using DNase to isolate RNA, electrophoresing RNA on an agarose gel, transferring separated RNA to a membrane, binding complementary probes to the RNA sequence of interest, and detecting the amount and size of the target RNA.
Analyzing the movement of RNA helps in understanding the reasons for different expression patterns in organisms and the control mechanisms based on growth stages.
Principles of Blotting Machine
The principles of blotting machines involve electrophoresis, transfer, blocking and antibody reaction, detection, and results analysis.
1. Electrophoresis
Samples containing biomolecules are moved in a gel under an electric field, separating them by size. Gels with varying compositions are used for DNA, RNA, and proteins.
2. Transfer
Molecules separated by electrophoresis are transferred to a membrane, replicating the separation pattern from the gel. The membrane and gel are alternately layered between filter paper and placed between two electrodes. When a voltage is applied, proteins move from the gel to the membrane following the electric current.
3. Blocking and Antibody Reaction
Blocking prevents nonspecific binding on the membrane’s surface during antibody reactions, reducing false detections. Antibody reactions involve introducing antibodies that specifically bind to the target biomolecule, visualizing the target molecule through labeled substances. These combinations enable the sensitive detection of biomolecules.
4. Detection
Detection involves labeled substances such as antibodies or probes binding to radioisotopes, resulting in the target molecule appearing as bright bands. Luminescence or color reactions occur, and the image obtained by detection devices or cameras is processed by analysis software to evaluate the presence, quantity, size, etc., of molecules.
5. Results Analysis
Results analysis processes the obtained images or data to evaluate biomolecules. It includes measuring the position and intensity of bands, background correction, creating standard curves, statistical analysis, biological interpretation, etc. This detailed analysis allows a comprehensive understanding of the presence, quantity, size, etc., of the target molecules.
How to Choose a Blotting Machine
When selecting a blotting device, consider the following points:
1. Purpose of Use
Select a device that meets the specific requirements for DNA, RNA, or protein analysis.
2. Sample Number and Size
Check the capacity for processing the number and size of samples based on the scale and target of your research. Larger DNA fragments or high-molecular-weight proteins may require devices capable of handling corresponding gel sizes.
3. Resolution and Sensitivity
Ensure that the device’s resolution and sensitivity meet the experiment’s requirements. High sensitivity is crucial for detecting low concentrations of target molecules. Resolution is influenced by gel concentration, transfer method, and detection technique, while sensitivity depends on the detection method, labeling substances used, and device design.
4. Transfer Method
Select a transfer method (semi-dry, turbo-blot, wet-blot, etc.) based on the efficient and uniform transfer of samples from the gel to the membrane. This choice is vital for the success of the blotting process.