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Printable Handouts
Navigable Slide Index
- Introduction
- Protein purification
- Total E. coli proteins - 2-Dimensional gel
- Typical protein purification scheme
- Purification summary table
- Features of proteins that enable various types of chromatography
- Peptide bond formation
- Oligopeptide with side chains
- 20 naturally-occurring amino acids
- Main types of molecular interactions
- Variables that affect molecular forces
- Protein properties - handles for fractionation (1)
- Protein properties - handles for fractionation (2)
- Protein properties - handles for fractionation (3)
- Protein properties - handles for fractionation (4)
- Separation processes that can be used to fractionate proteins
- Protein inactivation
- Proteases and protease inhibitors
- Protein oxidation and keeping proteins reduced
- Protein adsorption to surfaces
- Absorption to fluorescence reader plate wells diminished by low non-ionic detergent
- Destabilization and stabilization of proteins by salts
- A good all-purpose buffer that will make your protein happy
- What you can and can't learn from amino acid sequence
- General protein purification strategy
- Conclusions
Topics Covered
- Protein purification scheme and strategy
- Properties of proteins as handles for fractionation
- Obstacles during protein purifications and solutions
- Suitable buffers and practices to stabilize proteins
Talk Citation
Burgess, R.R. (2020, July 30). Fundamentals of protein chromatography: basic protein purification [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved November 10, 2024, from https://doi.org/10.69645/AWKT9237.Export Citation (RIS)
Publication History
Financial Disclosures
- There are no commercial/financial matters to disclose.
Fundamentals of protein chromatography: basic protein purification
Published on July 30, 2020
48 min
A selection of talks on Biochemistry
Transcript
Please wait while the transcript is being prepared...
0:00
Hello, this is Richard Burgess.
I'm a Professor Emeritus of Oncology in the University of Wisconsin-Madison.
I'll be speaking to you about the fundamentals of protein chromatography,
and in this first lecture on part 1,
the basics of protein purification.
0:20
In protein purification, the object is to
separate a particular protein from all the other proteins and cell components.
In E. coli, there are over 4,000 genes and in eukaryotes over 20,000 genes.
A given protein may be very non-abundant or it can be very
abundant and that will affect the amount of purification that is going to be needed.
The goal is not only to separate your protein from other proteins,
but from nucleic acids, from carbohydrates,
from lipids and from small molecules in the cell.
Enzymes, they can be soluble,
some are insoluble, some are bound to membranes,
some are bound to DNA,
they can be present in organelles,
in the cytoplasm, in the periplasm,
and in the nucleus region.
They can be secreted,
they can be post-translationally modified,
they can be processed.
1:12
The goal is to separate one protein from all the rest of the components in the cell.
Now, a way to illustrate this is to look at something called the
two-dimensional gel of E. coli cell extract.
The first dimension is what's called isoelectric focusing,
and you separate within a polyacrylamide gel.
You create a pH gradient that goes from about pH 4 to pH 10,
and then proteins within the gel when subject to an electric field,
they will move until they have no charge, that is, their isoelectric point.
Proteins vary in their isoelectric points from about 4.5 to over 10.
Once you've separated on the basis of isoelectric point,
you then lay that piece of gel onto a slab gel and run them on what's called an SDS gel,
which is a kind of an electrophoresis that separates on the basis of polypeptide size.
The size can vary from on the order of 160,000 in E. coli down to about 10,000.
When you do that and then run the gel and then stain it and then de-stain it,
you can visualize all the different proteins in the cell.
Notice that most of the proteins are in the region between about 160
and 10,000 and between about 4.8 and about 10 isoelectric point.
Suppose there is a protein that has an isoelectric point of 6 and
a molecular weight of 40 is one of those little spots in the middle of everything.
The goal is to separate that from all the others.
Now if you were to very high resolution
separate and get all the things that had exactly that isoelectric point,
and then on it's second step of a purification based on size,
get all the things that had a molecular weight of 40 kilodaltons,
your protein would be largely pure,
but very rarely can one do such a clean separation.
One has to use a series of fractionation steps to
successively remove components of the cell and other proteins.
You may note that in the lower left-hand corner of
the 2D gel are some large spots that are very basic.
They have isoelectric point of nine or 10 and are very
small between 10 and 15,000 daltons.
These are ribosomal proteins,
very abundant, very small,
very basic and most of the proteins you can see are
clustered in a slightly more acidic region of the 2D gel.