Instrumental Analytics Part I PDF

Instrumental Analytics Harald Hundsberger, Krems Wintersemester 23/24 INSA Overview Centrifugation UV/Vis-Spectroscopy and applications Measurements: Kinetic/Endpoint Fluorescence Spectroscopy & Applications Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, Lumin...

Instrumental Analytics Harald Hundsberger, Krems Wintersemester 23/24 INSA Overview Centrifugation UV/Vis-Spectroscopy and applications Measurements: Kinetic/Endpoint Fluorescence Spectroscopy & Applications Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, Luminescence Chromatografy Principles IEX FPLC HPLC Electrophoresis/CE 2D Electrophoresis Mass Spectroscopy  Instrumental Analytics Lecture deals with instruments but main focus is the application ! Flow Cytometry https://oncohemakey.com/principles-of-flow- cytometry/ Part-I Centrifugation Centrifugation- Types of Centrifuges Centrifugation is a wide spread technique All labs is industry and academia do have a lot of different centrifuges for different applications Benchtop C. low volumes and low speed Centrifuges with higher volumes (several liters) -big laboratory centrifuges Ultracentrifuges - Vacuum is applied (100.000 rpm) high g forces to seperate subcellular components. Elutriation Centrifuges, for cell separation (size) Safety Considerations UC Safety Considerations using Ultra Centrifuges (and other centrifuges) Use specified rotors only (user manual) Use specified sample holders only Weight samples and avoid unbalance (sub milligrams !!!). Rotors and other equipment sometimes have specified lifetime !!! DANGER OF LIFE !! Classification on Rotor Type Fixed angle rotors Sample Swing out rotor separation Centrifugation-Basics V= sedimentation speed g = relative centrifugal accelaration d = size of particle ρ(p)= density of particle ρ(m)= density of medium η = viscosity of medium V= d2(ρp-ρm)g/18η Svedberg Equation If centrifugation occurs in media with lower density of particles and low viscosity, the size is the dominating factor for sedimentation !! Centrifugation Techniques Differential Centrifugation Cell disruption Pellet nuclei Pellet mitochondria Pellet ER and other membranes Zonal Centrifugation Medium consists of a density gradient for example sucrose: density and viscosity are increasing Centrifugation is done with high g-forces and over longer time periods Gradients slows down fast particles maximum density of medium should be higher Recommended for particles than the lowest density of the separated particles which differ in size ! C. is stopped before particles will pellet Fraction collection Media for density Gradients Cesium Chloride Percoll advantage is low viscosity, disadvantage good osmolarity capabilities for whole organelle high osmolarity, used for nucleic acid preparations separation Sucrose Adv.: non-inionic, no interaction between biological material, Disadv.: low resolution  Applications for Centrifugation Pellet Cells, Harvest Cells Yeast, Bacteria other cells from fermentation Protein Purification Fractions of cells Purification of membrane proteins Desalting/Concentration of protein solutions (Amicon tubes) Pellet Nucleic Acids Plasmid Prep RNA Prep Spin columns (Silica based) Subcellular Fractionation Cell separation Elutriation Part II - Spectroscopy Basics Optical Spectroscopy Advantages: Little requirements on sample preparations, sample purity and work load Well defined instrumentation (Laboratory standard equipment) Applications for Spectroscopy in Biochemical Disciplines: Structure (CD-Circular Dichroism) Concentration (UV/Vis, Fluorecence) Bound/non bound state (Fluorescence Polarization) Reaction/Kinetcis (Fluorescence, Absorbance) Analysis of substances (Absorbance, IR-Spectroscopy) What is Light ? Electric & magnetic wave = Light ! Electric & magnetic vector are oscillating sinusuidally Dualism of light: light can act as particle: corpuscular theory Polarized Light Electro Magnetic Spectrum E=h.v E……………….Energy The higher v the more h………………..Planck`s constant energy light has: v………………...frequency UV light causes sunburn  Interaction of Light and Material/Sample Light can interact with material: photons can bring electrons to higher energy levels Transitions are very fast (S0 to S1 10-14 sec !) Light can be absorbed without later radiation Light can be absorbed and can cause later radiation Excited state has finite life time Excited state is more unstable/reactive fast:fluorescence slower:phosphorescence Part III - Spectroscopy UV/Vis/NIR-Spectroscopy Basics of Photometry Photometry: Measurement of the specific absorbence of light (absorbance at specific wavelength) Coloured liquids absorb a certain part of the wavelength spectrum Yellow colour means: blue light is absorbed Beer-Lambert law: relationship between absorbence and concentration of the absorbing substance.  Photometric Measurements Concentration of a solution can be determined with spectrophorometry if: pathlength (usally 1cm) molar extinction coefficiant (if not known, a standard curve is done) Absorbance is known (measured) https://www.youtube.com/watch?reload=9& v=BST5GRsAnPk Photometric Measurements Measurement unit for absorbance is OD (Optical Density) Modern Photometers display absorbance values in OD (optical density) What is OD ? A = -log T (Transmission) T = I/I0 I.....Light intensity after passing through the sample I0....initial light intensity Relationship Transmission and OD T=100% OD=0 I=100, I0=100; T= I/I0 = 100/100 = 1; A= -log T = log 1 = 0 T=10% OD=1 I=10, I0=100; T= I/I0 = 10/100 = 0.1; A= -log T = log 10 = 1 T=1% OD=2 T=0.1% OD=3 Classification of Photometric Devices Lightbeam Single beam/dual beam Wavelength selection Filters/Monochromator Filters:limited wavelengths Monochromator:continious spectrum Detector Single Photodiode Diode array Specifications Important Spec ! NIST Standard Cuvettes: National Institutes of Standards and Technology Applications UV/Vis-Spectroscopy Protein Quantitation: Biuret 570nm Bradford at 595nm Lowry at 750nm, 650nm or 540nm BCA 562nm Applications UV/Vis-Spectroscopy Protein Quantitation: Bradford at 595nm Lowry at 750nm, 650nm or 540nm BCA 562nm 31.01.2025 Fußzeile | Titel der Präsentation 30  Applications UV/Vis- Spectroscopy Absorption at 280nm (semi-quantitative) Absorption due to tryptophan (tyrosine) residues (fast) - disturbance by other substances with absorption at 280nm - absorption different for different proteins, due to variation in Trp content Rule of thumb: A280 = 1 corresponds to 0.5 – 2 mg.ml-1 protein Absorption at 205nm (semi-quantitative) Peptide bonds do absorb at 205 nm not dependent on protein composition little dynamic range (1-100µg) Applications UV/Vis-Spectroscopy Enzyme Kinetics Many Spectrophotometers do have evaluation software avaulble for Enzyme Kinetics Many enzyme kinetic reactions are measured at 340nm (NADH cofactor) Proteases: colorimetric substrates available Applications UV/Vis-Spectroscopy Applications UV/Vis-Spectroscopy DNA Quantification Protein Quantification Main application for microplate based photometers: ELISA !! Enzyme Linked Immune Sorbent Assay Measurement Modes Single Wavelength Measurement Done with cuvette photometers Blank measurement is done and value is subtracted form sample (necessary because cell itself scatters light) Application example: Optical density of a bacterial culture Measurement Modes Dual Wavelength Measurement: Usally done with microplate readers Second measurement can compensate for unspecific signals Reference wavlength for ELISA mostly between 620-650nm Application Example: Substrates for AP : pNPP (para-nitrophenyl phosphate): 405 nm aminoantipyrene, phenyl phosphate: 492 nm Reference value at 620nm is subtracted from 405 or 492nm. Part V –Circular Dichroism Spectroscopy  Circular Dichroism Spectroscopy Polarisation of Light Linear Polarisation Circular Polarisation Circular Dichroism, often abbreviated to "CD", is displayed when an optically active substance absorbs left or right handed circularly polarized light preferentially Circular Dichroism is the difference between the absorption of left and right handed circularly- polarised light and is measured as a function of wavelength This absorbance method works only with chiral molecules (optical active molecules).  Circular Dichroism Spectroscopy CD is measured as a quantity called mean residue ellipticity, whose units are degrees-cm2/dmol. Chiral or asymmetric molecules produce a CD spectrum because they absorb left and right handed polarised light to different extents and thus are considered to be "optically active". Biological macromolecules such as proteins and DNA are composed of optically active elements and because they can adopt different types of three-dimensional structures, each type of molecule produces a distinct CD spectra.  Circular Dichroism Spectroscopy Secondary structure analysis of proteins with CD: Scan from 190nm to 240nm is performed Helices, Sheets , Turns and Coils give different CD spectra Biosimilar Analytics „Rituximab“ 31.01.2025 Fußzeile | Titel der Präsentation 42 Induction Week Instrumental Analytics Part II Harald Hundsberger, Krems WS 23/24 INSA Overview Fluorescence Spectroscopy & Applications Fluorescence Intensity (RT-PCR, HTS, Sequencing, CE, Laser Scanning Microscopy, FACS) Time resolved fluorescence Fluorescence Polarization Fluorescence Life Time Luminescence Label Free Detection Plasmon Surface Resonsance Spectroscopy Fluorescence Fluorescence became to one of the most important detection technologies in the biomedical continuum Gene expression analysis of 40,000 genes in paralell ! Only with fluorescence detection (Microarray !) Instruments in labs using FI for detection and quantitation of molecules Important tool in tissue engineering (fluorescence labeled antibodies) Key technology in drug discovery (HTS- High throughput screening assays), Cell bases assays Fluorescence is moving more and more into the speciality testing market (BSE-testing, detection of genetically modified food…….) Key advantages of fluorescence: Sensitivity Specifity Multiplexing Part I: What is Fluorescence ? Fluorescence 1. 3 step process 2. Fluorophore is excited by/ absorbs light 3. The excited state exists for a finite time 4. Energy(light) is emitted returning the fluorophore to ground state Excitation and Emission Spectrum of fluorescein: Excitation at 485nm (peak) Emission at 535nm (peak) Distance between excitation and emission peak is called stoke´s shift Fluorescence detection devices have to separate excitation from emission light Excitation and Emission Excitation of a fluorophore at three different wavelengths (EX 1, EX 2, EX 3) does not change the emission profile but does produce variations in fluorescence emission intensity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum.  Fluorescence and Multiplexing Fluorescence detection of mixed species. Excitation (EX) in overlapping absorption bands A1 and A2 produces two fluorescent species with spectra E1 and E2. Optical filters isolate quantitative emission signals S1 and S2.  Fluorescence Dyes A lot of fluorescence dyes are available Can be coupled to biomolecules (proteins and antibodies) Different spectral properties Calcium sensing pH-sensing  Photobleaching Fluorescence process is rapid (10-8 seconds) and cyclical. The excited state is more chemical reactive than the ground state. When high power light sources are used (lasers), occurs also with lower energy light sources. Cyanine dyes are more resistant to photobleaching  Fluorescence Quenching Quenching causes decrease or loss of signals: – Energy of an excited dye is transmitted to another dye molecule – when samples are to labeled too densely, or if dye is too concentrated – Alternatively a quencher can be added – Fluorescence output dramatically decreased Part II: Hardware for Fluorscence Detection A Simple Fluorescence Detector Example: Microplate Reader (optics only), electronics and temperature control not shown ! Excitation light source Bandpass filters Alternative (double monochromator based Dichroic Mirror or Beamsplitter Lenses 1 light source 2+3+4 lenses Photomultiplier (CCD camera, photodiode 5 excitation/emission filters 6 beamsplitter 7 detector (Photomultiplier) 8 well of a microplate  Detectors for Fluorescence Photodiode Spectroscopy Spectrophotometer Photomultiplier Tube sensitive and high dynamic range Cuvette Fluorometers Microarray scanners Microplate readers FACS Flow Cytometer CCD - Cameras  Light Sources in Fluorescence LED`s light emitting diodes cheap but efficiancy can be high when high power LED´s are used (Light cycler from Roche, and DTX 880 from Beckman), can be pulsed ! Halogen lamp lower priced instruments Xenon flash lamp flexible because of wavelength range (200-> 1000nm) can be pulsed ! Lasers (argon, he/ne, nitrogen, laser diodes) Laser scanning microscopy FACS-fluorescence activated cell sorting Flow Cytometer High resolution scanners (microarray scanners) Part 3: Fluorescence Detection Methods and Applications  Fluorescence Detection Methods Fluorescence Intensity & FRET Time Resolved Fluorescence Fluorescence Polarization Fluorescence Lifetime Fluorescence Imgaging (Luminescence) Fluorescence Intensity Continuous light source (not pulsed) PMT collects emission light for a defined time period (integration time) Optical path for microplate reader fast measurements for 96, 384 or 1536 samples Fluorescence Intensity Applications High Throughput Screening assays Nucleic Acid and Protein quantitation Cell based assay Microarray FACS/Flow cytometry Quantitative PCR Confocal Laser Scanning Microscopy Flow Cytometry Flow cytometry Essential technology for cell characterization Expensive but a lot of informationfrom complex samples can be gained Cell sare characterized via fluorescence Cell are labled with antibodies or via intrinsic expression of fluorescence (GFP) Flow Cytometry Cytograms Expression of surface markers recognized by fluorescence labeled antibodies Clinical relevance: infected or not ? Detection of cells which could not be recognized by normal light microscopy methods Ratio calculation of subtypes of cells  Fluorescence Activated Cell Sorting If you want to separate a subpopulation of cells, you could do so by tagging those of interest with an antibody linked to a fluorescent dye. The antibody is bound to a protein that is uniquely expressed in the cells you want to separate. The laser light excites the dye which emits a color of light that is detected by the photomultiplier tube, or light detector. By collecting the information from the light (scatter and fluorescence) a computer can determine which cells are to be separated and collected. The final step is sorting the cells which is accomplished by electrical charge. The computer determines how the cells will be sorted before the drop forms at the end of the stream. As the drop forms, an electrical charge is applied to the stream and the newly formed drop will form with a charge. This charged drop is then deflected left or right by charged electrodes and into waiting sample tubes. Drops that contain no cells are sent into the waste tube. The end result is three tubes with pure subpopulations of cells. The number of cells is each tube is known and the level of fluorescence is also recorded for each cell.  FRET – Fluorescence Resonance Energy Transfer Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. Primary Conditions for FRET – Donor and acceptor molecules must be in close proximity (typically 10–100 Å). – The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor (see Figure). – Donor and acceptor transition dipole orientations must be approximately parallel.  Dual Color Optics for FRET Detection For multicolor analysis or FRET Both signals are separated simultanously FRET main application are HTS assays and imaging(microscopes) FRET-Imaging Localization of a protein interaction inside cells FRET assays are used for drug discovery see later: Time Resolved Fluorescence Assays  Real Time PCR (Quantitative PCR) Principle of quantitative PCR  Hardware and Application RT-PCR Most successful instruments in the life science market during the last 10 years ! Size decreased rapidly Today: LED based detectors Applications: RNA quantitation (Viral titer), Gene expression studies DNA quantitation from genetically modified food Nuleic Acid Quantitation with Fluorescence Superior performance over absorbance dsDNA ssDNA RNA Dyes also for protein available NanoOrange TM Cuvette based or microplate based Microarray & Scanners Analysis of 20,000-40,000 spots in parallel Each spot represents a single gene of an organism. Green means increase in expression, red means decrease in expression Very successful technology for research and pharma industry-drug discovery Focus Microarray in 5th semester- Biochemical Analysis Microarray Drug Discovery: – MA technology can provide useful info throughout the process of drug discovery. – Toxic properties of a drug can be monitored by analyzing expression profile induced by a drug candidate. – Looking for co expressed genes – https://www.youtube.com/watch?v=VN sThMNjKhM Confocal Optics Optics of confocal scanning microscope Difference Confocal non Confocal Drosophila embryo Stained with FITC (Fluoresceine) A non confocal B confocal Laser Scanning Microscopy Imaging of fast cellular events: Calcium influx Central second messenger Activity of mucles Fertilization Synaptic activity Apoptosis Differentitation Evolutionary conserved signal transduction pathway Calcium Imaging Ratiometric measurement Serves as internal control Important for screenings Special Measurement Modes FRAP-detection (Fluorescence Recovery After Photobleaching) Definition of the cell region to be bleached Brief illumination of the region with very high laser intensity Recording the progress of fluorescence recovery in the bleached area with high temporal resolution Changes in intensity in the bleached region represent the sum of all movements of the fluorescent molecules, whether passive (e.g., diffusion) or active (e.g., transport). The regeneration time (half-recovery period) is a measure of the speed of protein movement. Laser Scanning Microscopy Multiparameter Fluorescence: Spectral Detector of our microscope Time Resolved Fluorescence (TRF) TRF-Highly Specific & Sensitive Used Labels: Europium Chelates Europium Cryptates Samarium Dysprosium Terbium Hardware Requirements for TRF Pulsed Light Source Laser LED Xenon flash lamp High sensitivity Detection in the attomole range High specificity Background fluorescence is short lived TRF-FRET Assay TRF-FRET Assay Ratiometric read out for internal normalization Standard HTS assay in drug discovery Kinase, Phosphatase Molecular Interaction Luminescence Chemiluminescence:...is luminescence as the result of a chemical reaction. Bioluminescence:...is visible chemiluminescence from living organisms Source of Bioluminescence are Photoproteins Luminescencent ELISA Firefly Luciferase Photinus pyralis ATP dependent Light emission at 565 nm Luciferase  Luciferin  ATP  O2   Oxyluciferin  AMP  PPi  CO2  Light Luciferase  Luciferase Assays Firefly luciferase is by far the most commonly used bioluminescent reporter. This monomeric enzyme of 61kDa catalyzes a two-step oxidation reaction to yield light, usually in the green to yellow region, typically 550– 570nm Upon mixing with substrates, firefly luciferase produces an initial burst of light that decays over about 15 seconds to a low level of sustained luminescence Dual Luciferase Assay Reporter Plasmids Checking Promotors for strength or when they are induced Indentification of enhancer elements Aequorine For monitoring intarcellular Calcium BRET Bioluminescence resonance energy transfer Luminescence Detectors PMT based Injectors Cuvettes Microplates Imaging Systems Reporter Systems Induction Week Instrumental Analytics Part-3 Harald Hundsberger, Krems 20.9.2023 INSA Overview Chromatografy Principles IEX, Reverse Phase, HIC, HA, FPLC HPLC Electrophoresis/CE 2D Electrophoresis Chromatography Introduction into Chromatografic Separations Until the middle of the 20th century separations were largely carried out by classical methods such as Precipitation (example !) Distillation Extraction Today sample analysis (particular multicomponent samples) are carried out by Chromatografy Electrophoresis C. was invented by Russuin botanist mikhail Tswett Chlorophylls and xantophylls pigments from plants were seperated via small calcium carbinate particles Seperated species appeared as colours and therefore the name for the technique: Chroma (color) and graphein (writing) Chromatography Has grown rapidly during the last 50 years 12 Nobel Prizes from 1937 and 1972 were chromatography played an important role In Biomedical Continuum Liquid Chromatografy plays important role General Description of Chromatography In all chromatograhic methods, the sample is transported by a mobile phase, which may be a gas or a liquid Mobile phase is forced through stationary phase which is fixed in a column or in a solid surface Components of sample which are strongly retained by stationary phase are moving slower Components which are weakly held are moving faster Differences in mobility are causing zones or bands which can be analyzed qualitatively or quantitatively  Sensitive method for Gas Chromatography determination of gases and volatile substances Non reactive carrier gas is forced through a temperature controlled column Detection is done via flame ionisation Stationary phase: – Silicium Oxide Peptides and proteins cannot be vaporized without beeing destroyed Only important for carbohydrate and lipid analysis  Gas Chromatography Carrier gas for GC: – Nitrogen – Helium Capillary is heart of the system and is coated with different functional groups – typcical length is 15-20 meters Columns can be operated in the temperature range from RT to 300°C Cryogenic operation is also possible (-20°C to 20°C) GC detectors: – The compound and detector interact to generate a signal. The size of the signal corresponds to the amount the compound present in the sample. There are several different types of detectors that can be employed, depending on the compounds to be analyzed. These detectors can measure from 10-15 to 10-6 gram of a single component  Liquid Chromatography More suitable for biomolecules compared to GC Protein and Peptide separations (preparative) are mainly done with liquid chromatography systems (FPLC)  Components for Liquid Chromatography Pumps for transport of mobile phase and for creation of gradients and for controlled flow rates Valves controlling liquid stream column for separation process Detector (UV/VIS) Fraction collector Terminology – Chromatografy (Liquid Chromatography) Stationary Phase A column Filled with column Material Biopolymer Functional groups which interact with Analyte which is dissolved in mobile phase Peptide Protein Fraction collection Drops leaving the column are collected with fraction collector Terminology – Chromatografy (Liquid Chromatography) Mobile Phase: Buffer where analyte is dissolved Isocratic separation If analyte is not immobilized to stationary phase, no change of mobile phase during separation Gradient separation 2 pumps needed Composition of mobile phase is changed during separation process (salt concentration, pH, Detergent….) Linear gradient Step gradient Terminology Liquid Chromatography Retention time & volume: The retention time: time between the injection of a solute and the time of elution of the peak maximum of that solute. The retention volume is the volume of mobile phase passed through the column between the injection point and the peak maximum Terminology Liquid Chromatography Dead volume: Dead volume = Sample injection volume + volume from the tubing connecting the column to the injector and detector + volume from fittings, connectors, and pre- column filters + detector flow cell volume. Tubings with broad diameter !! Detectors for Liquid chromatography UV/Vis detector Protein and Peptide detection, DNA Fluorescence detector Proteins & Peptides Conductivity detector pH and Salt concentration Protein Purification 31.01.2025 Fußzeile | Titel der Präsentation 16 Introduction Why purify proteins ? Functional and/or structural studies Industrial or pharmaceutical applications Generate antibodies Determine proteine sequence Introduction Why purify proteins ? 31.01.2025 Fußzeile | Titel der Präsentation 18 Classification on Stationary Phase Functional Groups (For Biomolecules such as Proteins) Ion Exchange Chromatography Hydrophobic Interaction (HIC) Affinity Chromatography Size exclusion chromatography (Gelfiltration) Introduction Applications requiring pure proteins Introduction Comparison of expression systems Mammalian Characteristics E. coli Yeast Insect cells Cells Cell growth rapid rapid slow slow Compexity of medium minimum minimum complex complex Cost of medium low low high high Expression level high low-high low-high low-moderate secretion to secretion to secretion to secretion to Extracellular Expression periplasm medium medium medium Posttranslational Modifications only procaryotic often inclusion refolding maybe Protein folding bodies required proper folding proper folding simple, no sialic N-linked Glycosylation none high mannose acid complex O-linked Glycosylation no yes yes yes Phosphorylation no yes yes yes Acetylation no yes yes yes gamma carboxylation no no no yes Membrane Proteins Special types of proteins Parts of protein which are located inside a membrane are hydrophobic-special environment Glucose-6-phosphate transporter Purification process for membrane proteins differ completly from „soluble“ cytoplasmatic proteins. Integral Membrane Protein Exctration Integral membrane proteins require use of detergents for extraction and solubilization Detergents have same properties of lipids: Polar head group Hydrocarbon chain Hydrocarbon chain binds to hydrophobic part of protein-Protection against aggregation (Precipitation/aggregation) Detergent solubilized protein can be purified by conventional techniques (detergent needs Detergent micelle to be added to buffers !) Handling Proteins Proteins like to be: Kept on ice Fairly concentrated (µg to mg/ml) At right pH (not their pI ) Different buffer for storage and assay Avoid: Proteases Cyles of freeze thaw Factors affecting protein Stability Factor What can I do ? Temperature Avoid high temperatures, keep solutions on ice Determine effects on freezing, Include Glycerol in buffers. Store in Freeze thaw Aliquots Do not shake, vortex orstir vigorously (prtein solution should not Physical denaturation foam) Solution Effects Mimic cellular environment (neutral pH, ionic strength) Dilution Effects Maintain protein concentrations > 1mg/ml as much as possible Oxidation Include DTT or beta-Mercaptoethanol in buffers Heavy Metals Include EDTA in buffers Microbial Growth Use sterile solutions, include anti microbials Proteases Include protease inhibitors Protein Surface Typical surface of a momomeric, cytoplasmatic, soluble protein. Hydrophilic groups are shown with +/- symbols (interaction with water,solubility of protein) Hydrophobic patches of protein surface are grey shaded. Isoelectric Point (pI) The isoelectric point (pI) is the pH at which a protein carries no net electrical charge. Separation by Precipitation Precipitation at isoelectric point (pI) Important !: >pI all proteins have neg. net charge !