When beginning a new project,
where do you start? With a blank page, a bunch of hopes and good intentions? With a protein purification project whatever you do, before you begin, there are a couple of things
you always need to consider.
What’s your ultimate goal?
This is the one thing you need to be crystal-clear about: what do you want to accomplish at the end of the project? Do you want to characterize your protein using biophysical methods? Determine the structure using X-ray crystallography or NMR? Use your protein in a functional study? Raise antibodies in a rat or a mouse model? Whatever the ultimate goal, it will decide the quantity of protein needed, the purity and analysis techniques used and these will have a major impact on your planning.
For example with our DHFR project, we’re performing a detailed biophysical characterization including activity and binding studies.
But with any protein purification, there are four key aspects to consider:
1. Quantity
The quantity of protein needed varies enormously depending on goals. The scale can be from picograms (e.g. for mass spectrometry analysis) to kilograms (e.g. therapeutic proteins). The most common quantities that you will require within a research setting are in the µg to mg scale, for functional and structural studies.
2. Purity
Again the level of purity required for a protein is intrinsically linked to your goals. To be able to raise antibodies, purities of 90-95% are enough. For crystallography or characterization studies, purity of 99% is often required.
3. Activity
It is very important to decide whether the protein must be active after purification or not. Protein activity is not important in only a handful of applications; fundamental characterization studies, such as mass determination, amino-acid analysis, etc. Usually in today’s research, and as a rule of thumb, plan to retain the protein’s activity.
4. Homogeneity
Size or charge homogeneity depends on questions like: is your protein likely to aggregate? What are the consequences for your ultimate goal if it does aggregate? A common pitfall is that a very pure protein sample could be in the form of inactive aggregates; determining if this is acceptable again hinges on the final application.
Since our project includes assay development, we need to consider how much protein we have to purify before planning the protocol; so as to avoid low quantities of the protein in any further experiments. (From bitter experience :) many of us have learnt that an under estimate of how much protein you need, can lead to running out of protein half way through or before you have all the results you need. To avoid this, perhaps work in 10s of milligrams at least, as re-doing the experiment takes time and ultimately costs more than the increase in scale.) If you do run out, you may end up having to express and purify more protein and using different batches of purified proteins in the same experiments. This introduces another source of variance and uncertainty when you interpret your results.
So with our DHFR project we aim to purify 10mg quantities at a purity of >95 %, which would be more than enough to perform the activity and binding studies
A couple of tips when planning your project:
In our next post we'll look at what you need to know about your sample, but in the meantime if you want to share your own tips or have any questions, let us know using the comments/contact boxes below.
For more on how to simplify planning & execution of protein purification download our free handbook
What’s your ultimate goal?
This is the one thing you need to be crystal-clear about: what do you want to accomplish at the end of the project? Do you want to characterize your protein using biophysical methods? Determine the structure using X-ray crystallography or NMR? Use your protein in a functional study? Raise antibodies in a rat or a mouse model? Whatever the ultimate goal, it will decide the quantity of protein needed, the purity and analysis techniques used and these will have a major impact on your planning.
For example with our DHFR project, we’re performing a detailed biophysical characterization including activity and binding studies.
But with any protein purification, there are four key aspects to consider:
1. Quantity
The quantity of protein needed varies enormously depending on goals. The scale can be from picograms (e.g. for mass spectrometry analysis) to kilograms (e.g. therapeutic proteins). The most common quantities that you will require within a research setting are in the µg to mg scale, for functional and structural studies.
2. Purity
Again the level of purity required for a protein is intrinsically linked to your goals. To be able to raise antibodies, purities of 90-95% are enough. For crystallography or characterization studies, purity of 99% is often required.
3. Activity
It is very important to decide whether the protein must be active after purification or not. Protein activity is not important in only a handful of applications; fundamental characterization studies, such as mass determination, amino-acid analysis, etc. Usually in today’s research, and as a rule of thumb, plan to retain the protein’s activity.
4. Homogeneity
Size or charge homogeneity depends on questions like: is your protein likely to aggregate? What are the consequences for your ultimate goal if it does aggregate? A common pitfall is that a very pure protein sample could be in the form of inactive aggregates; determining if this is acceptable again hinges on the final application.
Fig: an
overview of how the scope of a project should be set depending
on application
Since our project includes assay development, we need to consider how much protein we have to purify before planning the protocol; so as to avoid low quantities of the protein in any further experiments. (From bitter experience :) many of us have learnt that an under estimate of how much protein you need, can lead to running out of protein half way through or before you have all the results you need. To avoid this, perhaps work in 10s of milligrams at least, as re-doing the experiment takes time and ultimately costs more than the increase in scale.) If you do run out, you may end up having to express and purify more protein and using different batches of purified proteins in the same experiments. This introduces another source of variance and uncertainty when you interpret your results.
So with our DHFR project we aim to purify 10mg quantities at a purity of >95 %, which would be more than enough to perform the activity and binding studies
A couple of tips when planning your project:
- Try to keep it simple and aim for the purity you need for your experiments, not above that
- Plan ahead, express and purify enough amount of protein from each batch to cover all experiments in the study
- Remember the more purification steps you use the more of the protein you will lose
In our next post we'll look at what you need to know about your sample, but in the meantime if you want to share your own tips or have any questions, let us know using the comments/contact boxes below.
For more on how to simplify planning & execution of protein purification download our free handbook
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