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Protein Quantification and Assessing Protein Purity
Biomolecules
University of Lincoln
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Protein Quantification and Assessing Protein Purity: SDS PAGE
Protein quantification
● Determining protein concentration ○ There are various techniques available to determine protein concentration: ■ Chromogenic assays e. Biuret assay ■ UV spectroscopy ■ Fluorescence spectroscopy
● ~ How much protein have you purified? ○ Why is this important? ■ Working out the specific activity of enzymes (enzyme assays) - important to know how much protein you have ■ Interrogating biological interaction (molar ratios) - how much protein/substrate will react with another ■ Setting up protein for crystallisation - need to know how many moles there are
● Biuret Assay ○ In an alkaline solution, Cu2+ forms a coordination complex with peptides containing 3 or more peptide bonds ○ This results in a reduction of Cu2+ to Cu+ and a colour change to a violet/blue solution which can be measured at 540nm - the colour change shows that there is protein present - usually at the top layer of the solution
● Folin-Lowry Assay ○ The Biuret assay is fast (10 minutes) but not particularly sensitive ○ Therefore, you can use the Folin-Lowry method - usually seen in older papers instead of recent papers ○ Firstly, Cu2+ is used as in the Biuret assay ○ Secondly, phosphomolydbate is reduced by tyrosine and tryptophan residues (aromatic residues) ○ This generates an intense blue colour that can be measured at 750nm ○ This method is more sensitive than the Biuret assay but takes longer and uses more reagents
● BCA assay ○ Uses the same copper reaction as in the Biuret assay ○ Then uses bicinchoninic acid (BCA) to detect the Cu+ formed ○ Two BCA molecules are chelated by one Cu+ resulting in a purple colour measured at 550nm ○ ~100x more sensitive than Biuret assay
● Bradford Assay - second choice
○ Uses an absorbance shift in Coomassie Brilliant Blue G-250 dye when bound to protein ■ Bound form has an absorbance maxima at 595nm (blue), unbound cationic form is green or red ○ Less susceptible to interference by other molecules ■ Detergents are still a problem ○ Short linear range (0-2000 mg/ml)
● Colourimetric assays summary
○ Many different methods available ○ Most based on the reduction of copper and associated colour changes ○ Choose the most appropriate one based on: ■ Time ■ Sensitivity ■ Expense ○ All use reagents to modify the proteins - destructive - will destroy proteins ○ Make sure you are within the linear range
● UV/visible light spectroscopy
○ UV light is in the 10-400nm range ○ Visible light is in the 400-700nm range
■ I0 = 2, I = 1
■ A = log10(2/1) = log10(2) = 0 (half of the light is absorbed)
○ Absorption of light is exponentially related to the: ■ Number of molecules in the solution (concentration) [c] ■ The lenght of the light path [l] ■ These are combined as the Beer-Lambert law: A =ecl ● E is the extinction coefficient ● Some units: ○ A is unitless ○ C has units of M (mol I-1) ○ I has units of cm ○ Therefore e has a unit of I mol-1 cm-1 or M-1 cm-
● Blanks
○ All of the assays involve measuring absorbance of a molecule ○ Other things in the solutions can also absorb ○ You need to make a blank ○ What should your blank contain? ■ Everything apart from the substance being measured which should be replaced by a suitable reagent/buffer ● Just need a buffer and protein ● The blank will be a buffer on its own
● Linear range
○ Assays are only accurate within the linear range ○ Choose correct standards - do all proteins react the same? ○ How many standards should you use? ○ Make each one separately from a stock? ○ Serial dilution?
● Visible light spectroscopy of proteins
○ Only useful for those that are coloured e. haemoglobin, GFP
● UV spectroscopy
○ A210 detects the protein backbone (peptide bond):
● As proteins all have peptide bonds the absorbance at 210nm is generally proportional to the size of the protein - backbone will absorb light here - cna measure the protein concentration here ○ Never used as other compounds will also be absorbed here ● Proteins absorb strongly at 210nm so this is sensitive ● Can be difficult to measure in practical terms - beward of other compounds that may absorb ● A280 detects certain amino acids: can measure the absorbance as long as it has there amino acids in
○ The presence of carbon rings generally leads to UV absorbance ○ Tryptophan > Tyrosine >> Phenylalanine ○ Would produce this graph: used the 280nm peak
● A spectrum
● UV spectroscopy
○ Is very fast ○ Doesn’t destroy your protein ○ The sensitivity depends upon the number of tryptophan, tyrosine and to a
● Fluorescence spectrophotometers
● Light source - comes through a window + is absorbed at 90 degrees of that ● Light passes through the excitation monochromator and slit - this determines the wavelength(s) of excitation ● Sample cell holds cuvette (2 windows at 90 degrees) ● Light passes through slits and emission monochromator (at 90 degrees to the excitation) - this determines the wavelengths detected ● Emission is detected, amplified and recorded
● Making fluorescence measurements
○ Excite at the maximal wavelength e. 494nm ○ Monitor emission (either single wavelength e. 521nm or spectrum) ○ Can change slit widths to alter power ○ Can quantify
● Fluorescence vs UV/Vis
○ Fluorescence has: ■ Enhanced sensitivity (up to 1000x) since the background is zero ■ Increased specificity - use of 2 wavelengths ○ However: ■ Fluorescence can be quenched due to interference with energy transfer or absorption of emitted light by other compounds present - loses its ability to fluores ■ Not all compounds are fluorescent - most compounds are not fluorescent
● Fluorescence summary
○ Absorbed energy is radiated as electromagnetic radiation e. light.
○ Fluorescent molecules tend to have certain structural features e. carbon rings. ○ Fluorescence can be easily detected using a fluorescence spectrophotometer. ○ Fluorescence can be used to determine protein concentration.
● Summary ○ There are various techniques available to determine protein concentration: ■ Chormogenic assays e. Biuret assay - use to calculate protein concentration - use bradford assay this isn’t good enough or if there is not aromatic amino acids as a 2nd choice ■ UV spectroscopy - 1st choice ■ Fluorescence spectroscopy
Assessing protein purity: SDS PAGE (Detection and Analysis)
● How pure is the protein you have purified? ○ SDS-PAGE ■ Electrophoretic theory ■ SDS gels in practice ■ Calibration of molecular weight markers ● Advanced PAGE techniques ○ Isoelectric focussing ○ 2D gel electrophoresis ○ Native gels ○ Zymograms
● Why do we need SDS PAGE? ○ As a tool in purification: ■ To show the purity of proteins for: enzymes assays, structural biology, biophysics, sequence determination ○ For identification purposes: ■ To separate proteins according to size and observe differences between samples
● The overall processX-ray crystallography
● The result
● Proteins must be stained to reveal their presence
● Proteins must be stained (and gel de-stained) to reveal their presence
● Coomassie staining
○ In acidic environments, Coosmassie dye binds to protein ○ Stain binds to positive amine groups (via sulphonic acid groups) and also via Van der Waals interactions ○ Gels are first fixed to dehydrate them ○ Stain is then added and incubated for ~1h ○ Gels are de-stained to remove excess staining
● Silver staining
○ When Coomassie staining is not sensitive enough you can use Silver stain. ○ Again, gels are fixed but then exposed to silver nitrate which likely reacts with basic and thiol groups. ○ Reduction of silver leaves black/brown deposits.
○ Sensitivity = time and expense.
● Calibration of molecular weight marker ○ To calculate the relative mobility (Rf)~ ■ Measure the distance each of the standards and your samples have travelled from the top of the separating gel. ■ Then dividing this distance by the distance travelled by the dye front.
● SDS-PAGE summary
○ Proteins are denatured by SDS and gain negative charge. ○ Proteins are separated by PAGE on the basis of size alone. ○ Gels are stained to reveal the presence of the proteins. ○ Protein size can be estimated in relation to molecular weight markers (proteins of known mass).
● SDS-PAGE gels
○ Sodium Dodecyl Sulfate (SDS; C12H25NaO4S):
■ Coats protein with negative charge.
○ Ammonium persulfate (APS; N2H8S2O8):
■ Source of free radicals. ■ Used as an initiator for gel formation.
○ TEMED (N, N, N', N'-tetramethylethylenediamine; C6H16N2).
■ Stabilizes free radicals and improves polymerization.
● SDS-PAGE gels - pouring a gel
● Precast gels - time = money
● Advanced PAGE techniques
○ The isoelectric point (pI), is the pH at which a particular molecule or surface carries no net electrical charge (NO SDS in this experiment) ○ Avoid excess of pH and high buffer concentration (typically 10 mM HEPES pH 8 for Q resin) ○ Isoelectric focussing
■ Relies on the pI of the protein
● Isoelectric focussing
○ Uses a medium with a pH gradient ○ Voltage applied ○ Proteins migrate to their pI ○ Allows separation of proteins based on charge
● Difference gel electrophoresis (DIGE)
○ Modification of 2D SDS-PAGE ○ Each sample is tagged with a different fluorophore, combined and run on a single gel ○ Analysed by fluorescence
● Identifying your proteins
○ Achieved via mass spec after cutting out of spot
● Protein mass spectrometry
● Protein complexes - native gels
○ Native or non-denaturing gels ○ Mobility depends on charge and hydrodynamic radius ○ Changes in size therefore affect mobility
■ Changes in charge due to chemical degradation (e. deamination) ■ Unfolded, ‘molten globule’, or other modified conformations ■ Oligomers and aggregates (both covalent and non-covalent) ■ Binding events (protein-protein or protein-ligand)
● Protein complexes - native gels
○ Electrophoretic mobility is difficult to predict ○ Proteins can be re-natured in some cases and used to show activity (Zymogram)
Protein Quantification and Assessing Protein Purity
Module: Biomolecules
University: University of Lincoln
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- More from:BiomoleculesUniversity of Lincoln33 Documents
