A protein isolation protocol aims to safely and efficiently separate a protein that you are interested in from a mixture. Isolation of proteins is done from mammalian, insect, plant, yeast, or bacteria samples.
Lysing cells for a protein isolation protocol
The first step of a protein isolation protocol that begins with cells requires lysing the cells to reveal their contents. There are several methods that can achieve this which range from chemical to physical perturbation of a cell membrane or wall. You can read more about cell lysis in the article: How to lyse cells.
Protein isolation methods
After cells are lysed, a protein purification method needs to be selected. We will discuss two popular methods further, affinity chromatography and magnetic bead separation.
Affinity Chromatography for protein isolation
Affinity chromatography utilizes molecular interactions to help your protein of interest bind to a scaffold while other contents of a mixture are eluted away. This scaffold is called an affinity column.
There are many types of columns that are pre-packed with standard purification molecules. A common example is a Nickel column. These columns are lined with nickel-NTA (nitrilotriacetic acid) which have a high capacity for binding histidine. Proteins that are going to be purified using this method are expressed with something called a “his-tag”, which is a multi-histidine chain which will bind specifically with Ni-NTA while other proteins will bind non-specifically or not at all.
A binding buffer will be used for this part of the protocol, something that will be gentle and keep you protein of interest bound while being strong enough to discourage non-specific binding of other proteins. When enough buffer has flowed through the column to get rid of anything that is not your protein of interest, you will start to elute your protein off the column. Elution buffer will be more drastic than the other buffers because it is meant to disrupt the interaction of your protein with the nickel. This buffer will likely have a more extreme low or high pH or high salt content.
General affinity chromatography protocol:
- Lyse cells
- Prepare affinity column (this can include packing the column with resin, or charging the column)
- Flow samples through affinity column with binding buffer
- Flow washing buffer affinity column to remove non-specific binding
- Flow elution buffer through affinity column to release protein-column binding and elute protein of interest
Magnetic Beads for protein isolation
Magnetic beads, also known as magnetic particles, are now commonly used for protein isolation protocols as well. Magnetic beads do not require columns or resin for purification and are easily performed in microcentrifuge tubes at the benchtop. Magnetic beads come precoated with Ni-NTA, which can specifically bind your his-tagged proteins. The general protocol is simple.
General magnetic bead protein isolation protocol:
- Mix pre-bound magnetic Ni-NTA beads with cell lysate. Allow beads to interact with the mixture in a laboratory tube with time and pipetting.
- Put the tube containing the mixture on a magnetic separator rack. The magnetic bead bound proteins will be drawn towards the magnetic, while everything else in the mixture will remain free in solution.
- Remove solution from the tube, and fill it with a wash buffer.
- Wash buffer can be removed and washing can be repeated as many times as necessary to get a clean sample.
- After washing steps a final storage or experimental buffer can be added to the sample tube.
- The tube can now be removed from the magnetic separator rack so that magnetic beads with protein can be resuspended into solution.
- An elution buffer can be used to elute your protein of interest from the magnetic beads
The benefits of this method are the simple steps and the possibility of easily scaling up the process using constant magnetic force separators. These modern magnetic separators allow you to safely and efficiently separate your molecules at different volumes, from a few ml at R&D scale to tens of litres in full scale purification processes. Lastly, the modern magnetic separator also has the ability to monitor the separation process in real-time, checking the batch-to-batch consistency of your purification process. This can greatly improve your protein isolation protocol by giving you the control for improving/optimizing the magnetic bead separation procedure.