Analytics Flashcards
Protein purification advantage and disadvantage
Ad- fast growth rate, cheap, transform bacteria from plasmid dna
Dis- proteins fold incorrectly, lacks some post transitional bodies
Protein purification
- Lyse bacteria cell walls without denaturing protein if Interest, freeze thawing, triton x non ionic detergent sonication
Purify protein from extract lysate
Differential solubility, Affinity chromatography, Size exclusion chromatography, Ion exchange chromatography, Hydrophobic interaction chromatography, Isoelectric Focusing
Can require multiple rounds of purification
Track protein in purification process
Track protein in purification process by western blotting - Primary antibody binds target protein & Secondary antibody with tag for detection allows visualisation
Protein assay - Bradford assay / Bicinchoninic acid (BCA) assay
in alkaline solution - proteins reduce Cu2+ to Cu1+
Cu1+ complexes with BCA (Purple → Darker purple = more protein)
Include a range of known protein concentrations - Allows the construction of a standard curve
Enrichment factor- protein purification
Yield (%) = (enzyme activity after purification step / enzyme activity in original sample) x 100
Enrichment factor = specific activity after purification step / specific activity in original sample
Aka purification factor
Dependent on - origin of the starting source.& efficiency of different steps.
Differential solubility
Initial purification step
Polar water molecules interact with hydrophilic regions of protein - increases protein solubility - Oxygen in water more electronegative
Anything affecting protein charge, structure or water interaction affects solubility
Ammonium sulphate precipitation - salting out with high salt concentration
Proteins fold - charged/ polar amino acids = hydrophilic protein surface
Uncharged hydrophobic amino acids hidden inside structure
Proteins are solubilised by hydrogen bonding with polar water molecules
Addition of high salt concentration = displacement of the water molecules & precipitation of the protein
Water binds with salt ions instead of proteins
Different proteins have different solubilities in aqueous solution
Ammonium sulphate - Highly water-soluble, cheap. No permanent denaturation
Salt Removal and Buffer Exchange
Salt may not need to be removed prior to next purification step – gel filtration chromatography and hydrophobic interaction chromatography
Dialysis
Sample is placed in a bag with semi-permeable membrane - permeability based on target protein
Pores too small to allow passage of your protein - but big enough to allow passage of salt ions
Several changes of buffer eventually remove the salt from your sample
Gel filtration - pores separates sample components based on size
Load dissolved protein (and salt) onto column – flush sample through with buffer
Small salt ions enter the pores of resin, whilst large proteins pass straight through (carried in the buffer)
Diafiltration - Pressure-driven filtration membrane
Salt passes through membrane BUT Protein is retained in sample
New buffer can be added and protein can also be concentrated
pH & protein solubility
Proteins have an overall charge - dictated by the presence of amino acid side chains that can gain or lose H+
overall protein charge changes with pH
Charged amino acids are hydrophilic – form hydrogen bonds with water, increasing protein solubility
Isoelectric point - pH where a protein has no net charge
least solubility due to lack of interaction with water molecules - precipitation
Heat denaturation
Heating = denature →exposes hydrophobic areas that bind each other, causing protein precipitation
Some proteins don’t unfold after heating- thermos table above 45 degrees
Affinity resin/matrix composed of an affinity molecule
bound to a solid support
e.g. Sepharose beads
Affinity matrix specifically recognises protein of interest - Protein may have specific tag
Beads can then be centrifuged and washed, removing unbound extract components (batch purification)
Purified target protein can then be eluted from beads
Affinity resin can also be packed into a column (‘column purification) for larger scale purifications
Add cell extract, then several wash steps and then elute target protein
Wash steps with increasing NaCl concentrations
His Tag purification
His-Tag purification
Bacterial expression vector for the production of a His-tagged protein
His-tags bind strongly to beads coated with nickel (Ni2+)
Often used for purification of proteins from bacteria
Epitope tags
often used for protein detection, affinity tags for purification
Consideration - potential functional effect of attaching a large tag
N-terminal or C-terminal
Can also tag proteins with biotin – binds strongly to streptavidin
GFP tags (big ~30kDa) - allow visualisation of proteins (and immunoprecipitation) using ‘GFP-TRAP’ (Chromotek)
Some tags can be removed after purification (e.g. enterokinase cuts after DDDDK in FLAG tag)
Gel filtration chromatography
separates proteins based on size
Add cell extract and allow to pass through column with buffer (mobile phase)
Collect multiple fractions over time – increasing volumes of buffer
Elution volume = the volume of buffer at which a particular protein exits the column
Monitor protein elution with UV absorbanc
Bigger protein = elute faster - gel filtration
You know which fractions (elution volume) correspond to a specific mass
Can load sample in high salt buffer - therefore can perform straight after protein precipitation
Protein of interest
After GFC, take a sample from each fraction and perform a Western blot for your proteins of interest
GFC resins - designed to have pores that allow separation of proteins within a particular mass range
Can check mass range of sample using SDS-PAGE, then choose resin
Column calibration
A range of protein standards are used to calibrate a column
Protein elution monitored by UV Abs and elution vol matched to mass
Each lane corresponds to a fraction collected during gel filtration
Protein complexes -
Factors Affecting Separation in GFC
Protein complexes - Complexes generally intact when proteins in native state
Factors Affecting Separation in GFC
Size/mass of protein - molecular radius, proportional to mass
Shape of protein (e.g. globular vs fibrous)
Length of column – longer columns give better separation
Amount of protein – too much protein can cause broad elution peaks
Resin material (e.g. pore size)
separates proteins based on charge
charge comes from ionisation of amino acid side chains
At physiological pH, Glu and Asp lose H+ (acidic side chains)
At physiological pH, Lys and Arg gain H+ (basic side chains)
7 amino acids have side chains that can be ionised - ion exchange Chrom
Aspartic acid (pKa 3.9)
Glutamic acid (pKa 4.3)
Tyrosine (pKa 10.1)
Cysteine (pKa 8.3)
Arginine (pKa 12.5)
Lysine (pKa 10.8)
Histidine (pKa 6)
pKa value = acid dissociation constant (pH at which 50% ionisation occurs)
At pH below pKa - side chain accepts H+ (protonated)
At pH above pKa - side chain loses H+ (deprotonated)
Isolectric point
Overall protein charge - determined by the proportion of acidic and basic amino acids
Isoelectric point (pl) - pH at which protein has no net charge
pH below isoelectric point = net positive – decreasing pH (protonation)
pH above isoelectric point = net negative – increasing pH (deprotonation)
If you know pI of protein, then you can adjust the pH to alter net protein charge
Exchange resin - IEC
Cation exchange resin - Binds to positively charged proteins (‘cations’)
Resin has a –’ve charge (e.g. CM-cellulose, S-Sepharose)
Anion exchange resin - Binds to negatively charged proteins (anions)
Resin has a +’ve charge (e.g. DEAE-Sepharose, Q-Sepharose)
To purify target protein - need to use appropriate buffer pH and the correct resin
Want target to bind to resin
Bound proteins are then eluted with buffer containing increasing salt concentration
Hydrophobic interaction chromatography
interaction between hydrophobic patches on protein and resin coated with hydrophobic material
In aqueous solution - HIC
In aqueous solution - proteins have hydrophilic surface with hydrophobic patches
water forms a ‘shield’ around the protein surface – hinders hydrophobic interactions
HIC - sample is prepared and loaded onto column in high salt buffer
Salt - displaces water and exposes hydrophobic patches
For protein binding to the resin, salt concentration is inversely proportional to protein
Hydrophobicity - HIC
For protein elution, a decreasing salt gradient is used.
Factors that may impact elution
Choice of salt in buffer (Hofmeister series)
Include non-ionic detergents (reduce hydrophobic interactions)
Reduce temperature
Change pH – proteins are least soluble (most hydrophobic) at their isoelectric point
Isolectric focusing
At a specific pH proteins have an overall neutral charge
Isoelectric focusing
Protein is loaded onto a gel with stable pH gradient
An electric field is then applied – proteins migrate based on their charge
Proteins will migrate along the pH gradient until they reach their isoelectric point
At high pH - protein is -’vely charged
At low pH - protein is +’vely charged
phosphorylation - adds a negative charge to proteins and alters migration in IEF
2-Dimensional Electrophoresis- protein analysis
First separation - based on charge (IEF) -follow with SDS-PAGE
Second separation - based on molecular weight (smallest move fastest)
Can perform Western blotting after 2D electrophoresis to detect protein of interest
SDS-PAGE protein analysis
Separates proteins based on their size
Need to unfold proteins (denature)
Use sodium dodecyl sulphate (-’ve charge) and DTT (reduces disulphide bonds) and heat to 95C 5min
Protein samples are loaded into wells of gel and electric current is applied
-’vely charged proteins migrate towards positive electrode
After electrophoresis, proteins can be visualised using Coomassie Blue
Large proteins remain near top, smaller proteins migrate to bottom - Need to include molecular weight marker on gel
Checking Expression and Purity of your Protein
Protein assay to measure protein concentration - then analyse purity using SDS-PAGE
Lysis buffer and wash steps can be modified to improve yield and purity of your target protein
Ireduce non-specific binding to your affinity resin
For protein binding - salt concentration is inversely proportional to protein hydrophobicity
For protein elution - decreasing salt gradient is used.
Factors that may cause low proteins concentration
Poor protein expression in bacteria – optimise growth/IPTG
Inefficient lysis – try other methods/combinations
Inefficient purification – reduce detergent/salt
Inefficient elution – optimise
Protein is insoluble – optimise expression conditions/use mammalian host
Protein degradation - proteins are prone to degradation throughout the process
Minimising Proteolysis - Major cause of protein degradation are protease enzymes released during cell lysis
Low temperature, Work quickly, protease inhibitors, chelators, SDS-PAGE
Factors that affect protein migration - purity factors
Proteins generally migrate based on mass - but can migrate based on size
Large post translational modification (e.g. ubiquitylation and glycosylation) cause proteins to migrate at higher mass
Small PTMs (e.g. phosphorylation) generally don’t affect protein migration
If proteins are not fully denatured, they might migrate as complexes (e.g. dimer)
Inefficient reduction of disulphide bonds
High content of basic amino acids can affect migration
Immunoprecipitation
precipitating a protein out of solution using a specific antibody to protein of interest
agarose beads coated with protein A/G which bind to antibodies with protein of interest
Beads insoluble and heavy - precipitate using centrifugation or magnetism
isotype control antibody (gold standard) - primary antibodies that lack specificity to the target -help differentiate non-specific background signal from specific antibody signal.
% input method = sample / input
Fold enrichment = sample / noise (control)
Co-immunoprecipitation (Co-IP) & RNA-immunoprecipitation (RIP
Use - Analyse protein–protein interactions
Sample preparation (non-ionic detergents - e.g., NP-40, Triton X-100)
Pre-clearing (just beads)
Antibody incubation (target antibody or isotype control antibody)
Precipitation of protein/protein complexes
Washing
Elution and analysis of precipitate (low pH or high salt solution)
Analysis - SDS-PAGE, Western blotting, Mass Spectrometry
RIP
Proteins bound to RNA
Use - Study the physical association between individual proteins and RNA molecules in vivo.
Classes
Native – used to identify RNAs directly bound by the protein and their abundance in the sample.
Cross-linked – used to precisely map the direct and indirect binding site of the RBP of interest to the RNA molecule.
Proteins bound to DNA-Chromatin-immunoprecipitation (ChIP)
Proteins bound to DNA
Use - Investigate regions of genome associated with a specific protein
Steps
Cross-link and harvest cells (Cross link DNA & protein) - Cross linking agent = formaldehyde
Cell lysis & chromatin fragmentation
Immunoprecipitation
Wash, elution and cross-link reversal (Remove DNA from protein)
DNA cleanup and analysis of DNA - PCR, qPCR, microarray, sequencing
Controls
Input DNA - A chromatin sample processed parallel to the other samples but lacks the IP step.
No Ab control - A chromatin sample processed parallel to the other samples but immunoprecipitated without specific antibody
Isotype Ab control - A chromatin sample processed parallel to the other samples and immunoprecipitated with an isotype Ab control (IgG or IgM)
Histone H3 antibody - A chromatin sample processed parallel to the other samples and immunoprecipitated with anti-H3 ab
