tutorial 3rd week
2nd year biochemists
2nd year Biochemistry
1. Reading material
Please take a look at some of the following resources. There is no need to read them all, but of course it’s not
- Voet & Voet, “Biochemistry”, chapter 5
- R.K. Scopes, “Protein Purification: Principles and Practice”
- Allan Jones, “Practical Skills in the Biomolecular Sciences”
- Notes from Prof Endicott’s lectures in Michaelmas term (see WebLearn for past lectures)
- Search for reviews in Methods in Enzymology
- The internet! ;-)
2. Techniques in protein purification
Can each of you please pick one topic from the ones below:
Ion Exchange Chromatography
Size Exclusion Chromatography
Methods to determine protein concentration
Methods to assess protein purity
Please prepare an overview about the topic of your choice. It should contain the most important aspects, but should
be no longer than one A4 page. During the tutorial, you will present this to your fellow students, so please bring
along 6 copies of it.
3. Develop your own purification strategy
Please prepare a practical protocol for the purification of the protein SLP-X. Indicate the main steps of purification
in a flow chart and describe each sub-step in more detail, explaining the method used and why you chose it. Ideally,
you would like to have ~10mg of pure (>95%) enzymatically active (!) protein at the end of the process.
what did you ﬁnd most helpful?
An enzyme, SLP-X, found in sheep liver has an interesting dehydrogenase activity that you would like to investigate
further. Your collaborators have done some initial characterization of SLP-X and found the following:
(Please note: not all of this information is necessarily useful!)
• SLP-X is a cytoplasmic protein expressed at ~2mg/kg in sheep liver and it has not been detected in any
other tissues tested. A local butcher has offered you a large amount of sheep liver.
• Your collaborators have raised a monoclonal antibody (IgG) directed against SLP-X. They have kindly
offered you 3 mg of the antibody.
• Your collaborators have worked out a simple and robust protocol for testing SLP-X enzymatic activity.
• On an SDS gel (and confirmed using Western-blot analysis) SLP-X runs as a single band of molecular
weight of approximately 30 kD
• SLP-X has a pI of 5.2.
• SLP-X has no disulfide bonds or cysteine residues.
• SLP-X has a single glycosylation site with high mannose content.
• SLP-X absorbs strongly at 280 nm.
• The enzyme activity of SLP-X is lost at high temperatures. Initial tests have suggested that the protein is
stable for months at 4oC but only minutes at 37oC.
1. general considerations / strategy
2. puriﬁcation techniques
- chromatography methods
- other methods
3. protein detection
4. assessment of purity
Purpose of Protein Puriﬁcation 1.
- increasing stability
- functional studies
- structural studies
- large scale production
Choosing a strategy: 1.
...characteristics of the protein
- primary sequence:
- oligomeric state
- modiﬁcations (glycosylation, phosphorylation..)
- interactions with co-factors, ions
- cytosolic, external matrix, membrane protein?
- expressed soluble/insoluble?
Choosing a strategy: 1.
...cloning / choosing the right vector
- express tagged/untagged?
- allow for afﬁnity chromatography
- possibly improve solubility
- potential “tags” to consider:
- maltose binding protein
(important for SEC, coefﬁcient:
SDS PAGE) good absorbance
sequence: inhibitors? temperature?
Protein puriﬁcation is a multi-step procedure:
Based on protein properties choose number and type of individual puriﬁcation steps
separation technique, i.e.
ion exchange chromatography
original sample size exclusion chromatography is protein pure?
reverse phase chromatography is protein active?
i.e. from cell extract,
tissue, blood, peptide
After each step assess protein purity and activity.
Purifying insoluble proteins:
- some proteins cannot be expressed solubly,
even after optimisation of expression procedure and vector
- does not have to be a disadvantage!
- over-expressed insoluble proteins form cellular aggregates = inclusion bodies
- not much puriﬁcation needed
- but refolding can be a problem
- potential procedure:
- over-expression of proteins
- cell disruption
- centrifugation: this time keep pellet, discard supernatant!
repeated washing of pellet & centrifugation (with buffer containing detergent to remove hydrophobic cell parts such as membranes)
isolated inclusion bodies: solubilise protein with high concentration of denaturing agent (urea, guanidine hydrochloride)
centrifugation (discard pellet)
further puriﬁcation (afﬁnity tags such as HIS-tags that work under denaturing conditions are a common ﬁrst step)
potential further polishing step
2. Principles of Chromatography
Throw it in the Rhine!
This is what the professor of analytical chemistry during my undergrad used to say.
It is a strange analogy, but it does work.. kind of!
Throw a mixture of rocks that you’d like to separate into the rhine. Depending on the speed with which the rhine ﬂows (the
mobile phase) and the rockiness of its bed (the stationary phase), your different sample components will travel different
distances. Let’s say you throw your sample into the Rhine at the Bodensee, component A will travel till Karlsruhe, B until Wiesbaden,
and C will make it all the way to the Nothern Sea!!!
2. Column Chromatography
Solid Phase / Stationary Phase
Mobile Phase Can be a gas (GC), a liquid (LPLC, FPLC, HPLC).
2. Main Chromatography Methods
Separation by speciﬁc binding
Ion Exchange Chromatography
Separation by charge
Size Exclusion Chromatography
Separation by size/shape
Reverse Phase Chromatography
Separation by hydrophobicity
...batch vs column/continuous ﬂow
2. Common Features of Chromatography
3. elute (either by retention time, or by competition)
2. Afﬁnity Chromatography
- separation by speciﬁc binding of the protein of interest to the column
- using an immobilised speciﬁc ligand for the protein of interest or its puriﬁcation tag
- this ligand is usually immobilised on an agarose bead matrix
- for example His, GST, HA, GFP, ... but also speciﬁc antibody against protein
- bind, wash, elute
- elution through addition of compound to mobile phase that competes with protein
for binding to stationary phase
- this compound may have to be removed from the eluate through additional puriﬁcation step
2. Ion Exchange Chromatography
- separation through binding of protein to column via electrostatic interaction
- Column matrix is either positive (=Anion Exchange Chromatography, to bind negative ions),
or negative (=Cation Exchange Chromatography, to bind positive ions)
- depending on pI of protein and buffer chose anion or cation exchange
- carefully chose a buffer which does not bind to column!
- protein elution through use of a gradient of increasing ionic strength to weaken binding of
protein to column
- Some Common Ion Exchange Matrices (and Buffers)
2. Size Exclusion Chromatography
- also called gel ﬁltration
- separation based on mw/size
- column bed often consists of agarose beads with pores of a certain size determining the
- as sample ﬂows through, small components diffuse in an out of pores, while larger components
cannot enter the pores and ﬂow right through
- proteins are sorted by size/shape, big proteins are eluted before small proteins.
size exclusion chromatography proﬁle
2. Reverse Phase Chromatography
- separation based on hydrophobic interaction of sample with column
- often used for peptides rather than large protein, as proteins can unfold when binding to column
- column bed is composed of carbon-hydrogen chains of different length, often attached via Si
- samples are eluted through increasing gradient of hydrophobic solvent (i.e. acetonitrile) to
compete sample off the column
- often followed by lyophilisation step to remove solvent
2. Other Puriﬁcation Methods
- Salt Precipitation:
By adding ammonium sulfate. Masks ionic interactions between proteins, proteins precipitate
due to hydrophobic interactions. Good for enzymes.
- Hydrophobic Precipitation:
Using solvents. Reduce the solubility of proteins by favouring protein-protein rather than
protein-solvent interactions. Danger of denaturation.
Exchanges every particle below a molecular weight cut-off by osmosis. Good for buffer
exchange or refolding.
Removes solvent to leave only solid particles. Typically used for peptides in organic solvents.
Separate proteins by molecular weight.
Methods to determine the protein concentration 3.
- Biuret essay:
Peptide bonds react with alkaline copper sulfate to form a purple compound which absorbs at
540nm. Sensitivity 1mg.
- Lowry assay:
Purple compound from Biuret assay reacts with Folin-Ciocalteu reagent (phosphomolybdate-
phosphotungstate) to form a blue compound which absorbs at 650nm. Sensitivity 5ug.
- Bradford assay:
Coomassie Brilliant blue binds to positively charged residues in protein. This shifts the
absorbance wavelength of the dye from 465 to 595nm. Sensitivity 1-20ug.
- Spectrophotometric assay:
Uses aromatic residues in protein sequence which absorb at 280nm. (also possible: peptide
bond absorbance at 214nm). Keep in mind that many other substances absorb in that area.
- Amino Acid composition analysis:
Destroy protein and measure amino acid content by HPLC.Very accurate method, useful to
determine extinction coefﬁcient (as opposed to prediction, which assumes random coil).
4. Assess Purity of Sample
- SDS PAGE Gel Electrophoresis:
Mix protein sample with loading buffer containing sodium dodecyl sulphate (SDS), a positively
charged solvent that binds all along the protein’s amino acid chain. A polyacrylamide gel then
separates sample components by size only, as mass to charge ratio is now constant.
Small proteins migrate faster than big ones.
- Mass Spectrometry: SDS-PAGE gel
Separate proteins by size. More in Fridays tutorial!!!
- Western Blot:
Assessment of activity. (Figure from PIERCE webpage)
lane1: molecular weight marker