Horizontal and Vertical Gel Electrophoresis
Horizontal Gel Electrophoresis
Researchers often use horizontal gel electrophoresis to separate DNA and RNA. Prior to separation, they load the DNA and RNA into the wells of an agarose gel in the electrophoresis chamber. The chamber is connected to cathode and anode, which electric field is created when there is power supply. Due to DNA and RNA are negatively charged, they migrate from a negative end to a positive end.
The agarose gel contains small pores which allow the migration of small molecules. Hence, once the electric field is applied, DNA and RNA start to migrate. The smaller the molecules, the easier for them to pass through the pores. As a result, smaller DNA and RNA would migrate further compare to the larger one.
Horizontal Gel System in LabMal
You can find out more information about horizontal gel system from Cleaver Scientific below:
- Smallest gel system in the range (64 samples) – multiSUB Mini
- Extended width of the gel system allows more sample to be resolved (100 samples) – multiSUB Midi
- Allow up to 210 samples to be resolved per gel – multiSUB Choice
- Suitable for a high number of samples (550 samples) – multiSUB Maxi
Vertical Gel Electrophoresis
Vertical gel electrophoresis is a more complex set-up compared to horizontal gel system. Researchers normally use this system to separate proteins instead of nucleic acids. However, before you separate proteins, you need to disrupt the quaternary structure of proteins into the linear strand. You can treat the protein with sodium dodecyl sulfate (SDS) to break the disulfide bond to get the denatured proteins.
Once the proteins become linear strands, you can use the vertical gel to separate the strands. Vertical gel electrophoresis utilizes the polyacrylamide gel, which has a smaller pore size compare to agarose gel. This is due to linear protein strands is smaller than DNA and RNA. Hence, a smaller pore size of polyacrylamide gel is needed to separate them.
Vertical gel electrophoresis contains stacking gel and resolving gel. The stacking gel concentrates proteins that are loaded into the well so that the proteins can start to migrate at the same time. After stacking, the resolution gel separate proteins based on the molecular size. The top chamber of this system contains the cathode while the bottom chamber contains the anode. Upon separation, the negatively charged linear strands of proteins migrate toward the anode (top to bottom).
Usually, the user pours the gel solution between a set of glass plates to form a very thin gel that is less than 2mm. In contrast to horizontal gel electrophoresis, the buffer can only flow through the gel in the vertical system. It allows the precise control of the voltage gradient during the process. When combined with polyacrylamide gel, it provides better separation and resolution. Vertical gel electrophoresis is usually chosen for separating protein due to the ease to prepare polyacrylamide gel vertically.
Vertical Gel System in LabMal
You can find out more information about vertical gel system from Cleaver Scientific below:
- Vertical gel system that runs a maximum of 4 gels within an hour – omniPAGE Mini
- Ideal for second-dimension electrophoresis – omniPAGE Maxi Plus
Difference Between Horizontal and Vertical Electrophoresis
|Usually used to separate nucleic acids (50 – 20,000 bp)|
Usually used to separate proteins (5 – 250,000kDa)
|Utilize agarose gel|
Utilize polyacrylamide gel
|Only 1 gel|
Two layer gels: stacking gel and resolving gel
|Run under native condition|
Run under denaturing condition
|Relatively easy to set up|
Relatively more difficult to set up
|Ethidium bromide staining for DNA|
Coomassie or silver staining for proteins
How to Produce Reliable Results?
Gel electrophoresis is all about optimizing the running conditions: voltage, choice of the buffer, pH value of buffer, dye, correct reference marker and more. All the aforementioned factors can affect the resolution of the outcome of gel electrophoresis.
Concentration of both agarose or polyacrylamide gel is important as they determine the pore size of the matrix. The sample size must be able to move through the pore size in order to produce a result. Aside from the pore size, voltage determines the speed of migration of the biological molecules provided that the charge of the molecules remains constant. Thus, you can manipulate the percentage of gel and voltage to get the optimal electrophoresis result.
Choosing an appropriate buffer according to your experiment design is also an important factor. Choice of the buffer does not only limit to the electrophoresis process, but applications afterwards are also an important consideration for choosing the right buffer.
When choosing the reference marker regardless of being nucleic acid or protein ladder, it is important to have the slightest idea of what product size you are expecting. Using a ladder that is out of the product range will make the estimation of product size difficult.
As for staining, there is very sensitive dye such as silver that can detect protein as low as 1ng. If the protein band contains the amount of protein that is too low, the Coomassie blue staining method may fail to detect the protein. As a result, you may get a false negative result. In conclusion, running conditions are important aspects of running gel electrophoresis.