lectures

1. lag phase

2. exponential phase

3. stationary phase

4. death phase

lag phase:

• no increase in cell number

• cells may be old and depleted of atp

• need to adapt to knew environment (new enzymes, ribosomes etc need to be synthesized)

ecponential phase

• cells are growing at maximum rate

stationary phase:

• grpwth ceases, total cell number constant (balance between division and death) due to nutrient limitation

death phase

decline in nr of viable cells due to nutrinet deprivation and builup of toxic wastes

not often used for bacterias, mostly animal cells

time consuming and pipetting errors

= cells in total * nr squares / volume of all squares (2*10^-5)

spread= spread across surface

pour = embedded within agar layer

dilute samples and are spreaded put then

error: not immediate answer (in different phase when starting to count)

the more cells the less lght hits the detector (the more is scattered)

• measure optical density

• need to know optical light which is scatteres to calculate nr of cells

• amount of scattering is proportional to concentration of cells at low turbidity levels

• OD600_ safe and not harmful for bacteria and know that no light will be absorbed at tis WL

1. certain gene from a species —> gene is cut and placed (dna fragment ist ORF) in vector (expression plasmid)

2. transformation (plasmid brought into bacterial cell

3. culitured and induced to express desired protein

4. AB resistance gene leads to only survival of bacteria with vector

so the fragment is put into the wrong direction

1. structural genes

   1. lac z

   2. lac y

   3. kac a

2. promotor (rna pol. binds on it) —> TATAA

3. terminator

4. regulator

5. operator

gene lac regulator gene is always on

• no lactose —> repressor protein binds on to operator, no lactose can be expressed as rna poolymerase is blocked from binding onto promotor

• lactose is used as carbon source

• if lactose binds on repressor protein —> inactive and wont bind to operator —> polymerase can bind to promotor —> transcribes lac genes

• 11 betha sheets and one alpha helic barrel in center

• a. victoria

• chromophore fluorophore located in center (formed when protein folds)

• excitation peak: 488 nm

• emission peak: 510 nm

• 25 kda

• betha barrel

• anthozoa

• excitation peak: 587 nm

• emission peak 610 nm

• 26.7 kda

• electron jumps to excoted state (S1) when absorbing energy from light/movement

• travel to lowest state and give energy in form of light/fluorescence to turn into groiund state (S0)

• absorbed ligt is always more energy than emitted light bc energy always gets lost

• abs: high energy, low wl

• emitted: low energy, high wl

• OD600—> low energy and will not excoite cells to S1, just scatter light

• multiple labelong experiments

• helo marking distinct protein populations

• technique for separation, purifixation and testing of compounds (separation of mixture into components)

1. mixture dissolved into fluid solvent = mobile phase

2. carries through system (column , plate, sheet, capillary tube) on which material is called the stationary physe

3. different compounts have different affinities to stationary phase and travel at different velocities in mobile fliud —> separate

analytic:

• establishs presence or measures relativ eproportion of analytes in mixture

• with smaller material amount

preparative:

• separates components of mixture for later use = purification

• components used posteriorly

1. gel filtration: mol. weight and shape

2. adsorption: VDW interactions

3. ionic exchange: charge

4. hydrophobic: solute hydrophobicity

5. reverse phase: solute hydrophobicity

6. isoelectric focus: isoelectrip point

7. affinity: affinity interaction

• size exclusion:

  • fractionate molecules and complexes into particular size range

  • remove all molecules larger than particular size

  • combination of both

1. calibrate with standard where size is known

2. molecules pass through stationary phase (bead pores)

3. rate is depending on size (small particles take longer as they move into all pores —> longer pathway)

4. elution volume of tested protein can be used to determine molecular weight from calibration curve

solid: has pores

liquid: move with compounds but some bind to solid phase

pro:

• easy to use and interpret

• applicaple to all type of biomolecules

• applied and soluted in any solution

• separation independent pn eluent compostion

con:

• low resolution (compared to gradient elution techniques)

• leading capacities limited with respect to sample volume

• desalting of proteins of low mol weight

• mol weight estimation

• vaccine purification

• clean up step in purification scheme

• separating based on net charge

• covalemtly bound to solid support (pos or neg functional groups)

• charged compounds absorped and retained by ion exchanger having opposite charge wheras compounds that are neurral or same charge as media pass through the void

• binding compound is reversible —> adsorbed compounds are eluted with salt/ph gradient

• ! total change of protein is functional of ph solution

1. startng conditions: equilibration and binding solutes to be purified

2. adsorption of sample

3. start of desorption

4. end of desorption (elution, displacing ions)

5. regenration

anionic exchanger: positive: attracs negative charged analyts

cationic exchanger: negative: attracs positive charged analytes

1. change ph (decrease until gfp bekomes positive)

   1. not common: damages molecules and buffer may change uncontrollably

2. add salt (destroys electrostatic interaction)—> increasing ionic strength of eluion buffer

   1. more common, salt compound takes up gfp place

Isoelectric point:

• point of ph were no net charge occurs (all charged groups are on surface of protein)

• pH < pI = pos. charged protein, bind to cation exchanger

• pH > PI = neg charged protein, bind to anion exchanger

• pH =PI = no net charge

pro:

• high capacity and resolution

• concentration step

• sample volume may be large compared to column vloume

cons:

• sample eluted in high salt concentration —> desalting for further use

• solution must be low ionic strength and specific ph

• interaction btw hydrophobic proteins and HIC resins is influenced by running buffer

  • high salt conc: enhances interaction

  • low weakens

• separates proteins according to differences in their surface hydrophobicity

• start with high salt to üromote hydrophobic interaction—> then decrease —> more hydrophobic molecules bind stronger

• (protein with lowest hydrophobicity elute first (when low ionic strength, bc interactoin is reversed then))

1. equilibration (buffer a is pumped through column)

2. sample application and wash (sample is adsorbed/components washed out)

3. gradient elution: adsorbed samples are eluted in order of increasing hydrophobicity

4. regeneration: still remaining hydrophobic contanimants are washed out

pro:

• sample volume may be large compared to column volume

• concentration step

• high capacit and resolution

con:

• desaltinng required

• liquid chr. that involves separation based on hydrophobic interactions

• solid phase is less polar than mobile phase (normally mobile phase is non polar)

• —> hydrophobic surface in stationary phase

• less polar componnts have stronger affinity to non polar stationary phase

• —> more polar components are washed put earlier

• eluted by increasing concentration of organic solvent (acetone, ethanol) whic will increase hydrophobicity of liquid phase and molecules wont attach

• used for peptides and not proteins

1. equilibration

2. sample application

3. elution steps (1-3) gradient elution and organic solvents at end

4. washing (high organic modifier concentration)

pro:

• no sample volume restriction when using gradient elution

• concentration step

• high resolution

• used in most sample conditions

• salting step

con:

• use of organic solvent may denaturating protein (dangerous and expensive)

• careful pretreatment for some samples

• high purity of chemicals needed

• low capacity (risk of unfolding?)

• limited choice of eluents

• pattern difficult to interpret

• separating biochemical mixtures based on highly spec interation

  • ab and ag

  • enzyme substrate

  • receptor ligand

• the larger molecules, the stronger interaction

• IMAC: immobilized metal ac

  • his-tag proteins (metal immobilized)

  • with histidine tags with n or c terminus bind strongly to metal —> hexa his tag introduced to gene since not naturally in protein (put a tag and protein binds to metal)

  • target tag nect to other tag so that protease can cut and leave protein (mostly larger than his tag)

  • pH chnage to elute protein or adding competetice molecule (imidazole) to compete with metal

• mcherry:

  • high affinity to nickel: will dilute mcherry

• inroganic compounds

• polysaccharides

• synthetoc material

• physical properties (mech. strength, max flow)

• chromatographic properties (capacitiy, selectivity and resolution)

  
  

motion of dispersed particles relative to fluid under uniform electrical field influence

• particles carry el charg eon surface

• external el field is applied to change particles —> electrostatic coulomb force

surface charges surrounded by diffusive ion layer

• layer has same charge as surface but opposite sign

• formed due to interaction with surroiunding ions in fluid

• layer reduces direct interacton btw. surface charges and external environment

• force on ions in layer acts in opposide direction as external field excerts force on surface charge and ions on diffusive layer

• ds dna: long rods mugration relative to size

• ss dna: fold up—> migrate based on tertiary structure

  • agents added which disrupt hydrogen bonds to denature nucleic acids to behave like long rods (sodium hydroxide and formamide)

precast dye: run gel with dye —> öess dye loss

post-stain dye: dye after running

• sodium dodecyl sulfate

• polyacrylamide gel electrophoresis

—> unfolds an coats protein with negative charge in proportion to size —> mobility just on mol weight

smaller proteins move through easier/faster

• mostly analytical as proteins are unfolded

• stacking gel: lower con acrylamide gel

  • concentrates and focus protein inro tigh band before entering resolving gel

• resolving gel: separates proteins based on their size —> higher conc of acryl. gel

  • sieving effect due to smaller pore sizes

• both improve resolution and accuracy in determining mol weight on proteins

• 
  

1. silver stain (limit 1-10ng protein)

2. coomassie brilliant blue ( 50-100 ng)

• stacking/stacking : 5%

• resolving/running: 20%

• high gel = higher friction = lower movement

• loading buffer

• sample

• dye

• sds

• dtt

• glycerol

• start at different well and therefore ends in differen areas —> large band

• 2 gels need to be prepared

semi-quantitative (not precise, only increase/decrease seen)

to detect and analyze proteins (identify in many others)

no dye

• separation of proteins —> transferring on membrane —> AB to detect

1. protein separation (SDS Page)

2. transfer to membrane (blotting)

3. blocking -> to prevent nonspecific bindnig (adding solution that covers remaining spaces on membrane)

4. primary AB incubation (specific to protein of interest) —> bnds to target protein

5. washing: excess unbound prim ab to reduce background noise

6. sec ab incubation: recognizes prim ab (is linked to detection molecule (enzyme or fluorophore)

7. detection: detection on sec ab produces signal indicating target protein presence (fluor. or chemiluminesc.)

8. analysis: bands corresponding to presence of specific proteom

if strong interaction= high ka concentration componants is lower than binding compound

high affinita ka > 107 M-1

ka = [Ab-Ag]/[Ab][Ag]

target single position in antigen

1. inject antien in organism

2. organism creates ab against antigen

ab mixture binding to different parts of antigen (same ag but different positions)

• extract spleen of organisms injected with antigen

• isolate single cell

• create hybridoma (spleen cells bonded with cancerous cell to immortalize spleen cell)

• get culture of twin cells creating stable cell line that produces single type of AB

enzyme linked immunosorbent assay (quantitative)

• use AB and color change to identify a substance

• solid phase enzyme immunoassay (EIA) to detect presence of substance (ag) in liwuid or wet sample

• ag attached to surface —> sp. ab applied over surface to bing (ab linked on enzyme)

• substrate containing enzyme is added —> subsequent reacton produces detectable signal, most commonly color change

dorect assay: prim ab has enzyme

indirect assay: sec ab has enzyme

• 
  

• simplification: avoid expensive process of creating enzyme linked ab for every ag —-> using enzyme linked ab which binds to fc of other ab —> just need to label one ab instead of a lot prim ab

• signal amplification: more than one sec ab can bind to prim ap —>more signal for gene and more ab to detect

1. colorimetric (enzyme reacts with substrate —> colorchange) (spectrophotometer)

2. chemiluminescence —> emitted light (luminometer) —> high sensitivity

3. fluorescence (fluoroescence reader) —> high sensitivity

sandwich elisa

competitive

quantitative

• ag captured between 2 AB

  • 1 immobilized on plate

  • 2 linked to enzyme

• enzyme reaction produces measurable signal

• use purifies specialized ab to attach ag eliminate the need to purify ag before —> simplifying essay and high specifity and sensitivity

• more specific sinse ag binds to two ab (lower falsification)

1. unlabeled AB is incubated in presence of its ag

2. complexs (AB/AG) are added to antigen coating well

3. plate washed —> unbound ab are removed

—> if high complex concentration = low ab available to bind to antigen well

• if all AB saturated ag, no ab available to make light signal

• if singal: no ag or less ag attached

qualitative elisa: positive or negative

quantitative: OD of fl. units are interpolated into standard curve —> serial dilution of target

sandwich elisa

1. free monoclonal ab sp. for hCG

2. capture monoclonal AB bound to substrate (immobolized ab)

3. sandwich formed by combination of capt ab and free ab when hcg is present —> creating coloiur change

4. also control line: contains ab that always bind

• indirect assay to detect prim ab against AB against HIV in blood serum

• serum diluted and applied on plate where HIV ag are attached

• if HIV ab present in serum it binds to ag

• plate is washed to remove other serum components

• sec ab applied on plate linked to enzyme

• washed again

• 
  

sandwich elisa —> want to test whether you have ab against covid

• well is coated with sp. cov ag

• sample is added to elisa plate and ab bind to it

• sec ab added which has enzyme attached

• sec ab bind to prim ab

• substrate is added which binds to enzyme and color change seen

protein expression and phenotype live cells

• single cell analysation: size, shape and properties

• divide cells into individual populations

• cell surface can be detected = surface markers and cluster differentiation

• shows heterogeneity of cells : small nr of cells and are subsets

1. forwars scatter: cell size

   1. detected by detector on far side of cell from laser

2. side scatter: celll granularity

   1. detector located perpendicular to pass of laser beam

3. fluorescent emitted

• flow cytometry: quantify exact cell nr expressing specific protein and amount of proteins

• western blot: one band detected whose intensity depend on number of cells expressing specific protein

• polychromatic:

• Over 5 markers simultaneously detected

• Requires bandlass filter

• spectral:

• Full spectrum and allows large number of fluor. iPhone

• No filter required

1. Dot plots: 2 parameter (cell size and granularity)

2. Histogram: 1 parameter (nr cells ca fluorescence)

3. Density plots: 2 parameter (accumulation and relative densities

Fluorescence activated cell sorting

Process of separation Love cell populations into sub populations based on fluor. ich time associated with detection ab

To make sure they annealing is higher than annealing if strands again

Forward primer;

• always for antisense strain

• Is from 5’-> 3’

Reverse primer:

• on sense chain/ target

• Is from 5’-> 3’

General:

• sample chain is always 5’->3’, so the reverse primer docks in it from the end to the front

1. Taqman

2. Molecular Beacon

3. Sybr green

Quenching dye disrupts observable signal from Reporter dye when In short distance

• taqman probe (and primer) binds to target dna and taq polymerase completes strand

• Reporter dye releases (as well as quenching) from extending ds dna and therefore emits light as in excited state as quenching and reporter and not sticj with each other anymore

• The more denaturing and annealing the more emitted light

Hairpin dna confirmation with reporter and quenching dye at end (bc near they don’t show emission light as absorbed by quencher

If anneals with pcr product; no fluorescence blocked

• the more pcr product the stronger fluorescence

• dye intercalates into newly synthesized dna

• Cost effective and no specific probe for each target (can also get signal from other sequences)

Primary:

• directly isolated from Living tissue

• Not altered transformed

• Limited ability to divide and proliferate

Continuous:

• immortalized cells: modified/ adapted and have extended life span through immortalization bc of genetic modifications and transformations

• Divide continuously without reaching state of senescence -> long term Culture

N(t) = No * 2 ^(t/Td)

Growth surface covered by cells

Important as it affect cell behavior signaling and health

Adherent:

• attached to solid/semi solid substrate

• Growth limited by surface area

• Normal cells except hematopoietic cells

Suspension:

• floating in culture medium

• Limited by concentration of cells

• Agitation needed (shaking/stirring)

• Hematopoietic or transformed tumor cells, less common

1. trypsinization (enzyme that cleaves peptide bonds and detach cells)

2. Enzyme free solluction: accutase: chelating agents disrupt adhesion

3. Scraper/ lifter

• spontaneous or induced permanent phenotypic change (not nessecarly involve genetic material uptake -> difference to transformation)

1. Liposomes:

   • fuse with cell membrane and releasing genetic material

   • Integrate in genome Or remain episomal

   • Check by adding GFP or ab to inserted gene

2. Calcium Phosphate

   • forms precipitate with dna

   • Taken up by cell through endocytosis

   • Acidic environment in vesikel dissolute a complex and dna is released

3. Electroporation

   • electric field creates temporary pores in cell membrane allowing entry for foreign genetic material

   • High efficiency

4. Viral transduction:

   • Virus genetically modified

   • taken up through endocytosus

   • Viral capsule removed to release genome and then transcribed and translated by host cell

   • If rna virus, reverse transcriptase needed to be included

Combines pcr and reverse transcription

• amplify and quantify rna molecules

• Done because important to know what gene exactly expressed

• GFP intro transgene

• Resistant markers (Ab or drug resistant)

Ability to distinguish 2 objects close to each other

Rayleigh: 516 nm=0.5 microm

With oil: 239 nm

Abbe: 423 nm = 0.4 micrm

With oil 196 nm

• image formed based on scattered light

• Cell structures that scatter more light will be visible whereas background scatters no light and will be dark

• Light goes outside organisms but will be captured by organism when there

• 

• cannot use Lende of highest numerical aperture and any dirt or dust degrades image

• phase difference on light passing through different organisms part

• Variation in brightness against gray background

• Phase different at 1/4 lamda (need thin and uniform species)

• polarized light splits into 2 beams

Differential interference contrast

• interference of light passing through organisms

• Utilizes polarized light source and set of wollasten and nomaski prisms to create 3d like appearance (accentuating differences in refractive index within specimen)

• Not much contrast in thin objects

• Good for Protozoa and small organisms

Always excitation energy gets lost and therefore

Fluorescence (emission) always at lower energy/ higher wavelength than absorption

1. quantum yield (fl efficiency) calc: photons/ absorbed photons

2. Extinction coefficient: efficiency of capturing light (less critical)

1. Transmitted illumination

   • light which has passed through specimen

   • Reaches eye/ objective lenses after interacting with specimen

   • Not often used as high background (high unblocked excitation light)

2. Incident light

• light falling into specimen

• Epifl.

• Dichroic mirror: beam splitter: reflects light with shorter wl (excitation) and transmits higher wavelength (fluorescence/ emission)

• Unwanted excitation light deflects of observation light path

Mercury lamp

Xenon arc lamp

• short pass filter: absorb longer wl, passing shorter wl (high energy like excitation)

• Long pass: absorb shorter, passing longer wl (low energy like fluorescence.emission)

• Band pass: optical filter defining specific range

• photon absorbed = energy should match required energy for electronic transition

• Boosts electron to higher energy state (excited state S1) by absorbing light/ movement

• It can return to ground state s0 by at first reaching lowest level of s1

• Must then loose energy through heat release or emitting Photons like Fluorescence to drop to s0

• scans beam of light to get 3D image

• If sample Thick or out of focus it reduces background contrast

• 2 coincident focal points:

• 1. Illumination focal Point: laser light tightly focus onto specimen

• Detection focal point: rejecting information from outside the plane of focus

• high speed confocal microscope: scan more than one point at a time

• Fe tandem scanning (spinning disc): multiple pinholes to rapidly capture 3d image

Fluorescence resonance energy transfer

• phenomenon in which energy is transformed between 2 closely fluorophores without photon exchange

• Donator and acceptor

• Need to be 1-10nm close

• Proteins more stable in higher pH therefore it’s better to be charged negatively

• Cationic mostly with proteins with a general net positive charge

Larger ones are faster as smaller ones sieve throug gel pores and therefore take longer time

• anionic: differated by net surface charge

• Highly efficient

• Cost effective

• Affinity: more specific and efficient with higher yield

• Separated by specific interactions

Western blot

• protein transferred from gel to solid membrane

• GFP ab Leads to GFP binding

• Washing to get rid of rest

• Secondary ab added with linked enzyme

• Substrate is added generating color when secondary ab, protein and primary ab are in a complex to detect target protein

1. water (dilute concentration)

2. Buffer (hood working milieu)

3. dNTPs (to make strains)

4. Polymerase (enzyme which is making strains)

5. Primer (forward and reverse) docking in strand and polymerase starts from there

1. denaturation

2. Cycle of 30 (denaturation, annealing, elongation)

3. Final elongation

1. linearize Vector with restriction enzymes to produce sticky ends

2. Purify vector through gel electrophoresis

3. Purify amplified dna from pcr

4. Vector and dna are joined (sticky ends)

5. Perform transformation into bacterial cell

6. Selective agar with ab

7. Colony pcr using vector specific primers to confirm inserted dna

8. Western blotting to analyze expression

Nr of cells x volume of chamber x 2 x 7 ml

1. ph drop

2. High cell concentration

3. Cells rounding up of cell detachment from substrate

4. Cell Type and morphology

Monoclonal: bind only on one epitope on antigen

Polyclonal; can bind on different epitopes of antigen