isolation and analysis of biomacromoleculesold-biomikro.vscht.cz/vyuka/ibe/1_lecture_2015.pdfsalting...
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Isolation and Analysis of Biomacromolecules doc. Ing. Petra Lipovová, PhD.
http://biomikro.vscht.cz/vyuka/?Predmet=ibe • 7.10. 2015 Introduction - source of proteins, lysis, filtration, precipitation • 14.10.2015 Centrifugation, isolation of nucleic acid • 21.10.2015 Chromatographic techniques • 4.11.2015 Lecture canceled • 11.11.2015 Affinity chromatography • 18.11.2015 Recombination proteins • 25.11.2015 Work with program ProtNlab • 2.12.2015 Electrophoretic methods • 9.12.2015 Immunomethods • 16.12.2015 1st term of exam
Introduction source of proteins lysis of cells membrane process precipitation
1. to find suitable source of protein 2. lysis of cells 3. purification
Basic isolation steps
The source of proteins
microorganisms animal tissue plant tissue body fluid
Microorganisms
Advantages: Bacteria, yeast, fungi, algea
can be easily obtained in sufficient quantity (cultivation in fermentor...)
o selection of mutants with the required properties thermophilic organisms (production of enzymes with higher thermal stability)
o psychrophilic organisms gene engineering
Animal sources • Laboratory animals- mice, rats, rabbits • Human-body fluid (blood - plasmin, thrombin; urine -urokinase...)
• Invertebrates- insects, snails, molluscs
• Tissue culture - CHO
Plant Spinach, beets, peas, Arabidopsis thaliana, tobacco Disadvantage: the problematic growth under defined conditions plant suspension cultures
Caenorhabditis elegans
•ATCC (American Type Culture Collection) http://www.lgcpromochem-atcc.com/ •CCM (Czech Collection of Microorganisms) www.sci.muni.cz/ccm/ •FDA (Food and Drug Administration) (2001), Partial list of microorganisms and microbial-derived ingredients that are used in foods. www.cfsan.fda.gov/~dms/opa-micr.html •AMFEP (The Association of Manufacturers and Formulators of Enzyme Products) www.amfep.org • addgene.com
Where to get information about potential sources?
Your task is to isolate urease
Urease (EC 3.5.1.5) (NH2)2CO + H2O → CO2 + 2NH3
Helicobacter Pylori Urease drawn from PDB 1E9Z
http://www.madrimasd.org/blogs/microbiologia/wp-content/blogs.dir/110/files/1431/o_helicobacter.jpg
ATCC (American Type Culture Collection) http://www.lgcpromochem-atcc.com/
Plant Seed
ATCC® Number:
75043™ Price: £190.00; €285.00
Organism: Arabidopsis thaliana Heinhold
Designations:
S8-1-2A
Depositors: D Dennis, Y Poirier, CR Somerville
Biosafety Level:
1 Shipped:
dried, package of 25 seeds each order
Permits/Forms:
In addition to the MTA mentioned above, other ATCC and/or regulatory permits may be required for the transfer of this ATCC material. Anyone purchasing ATCC material is ultimately responsible for obtaining the permits. Please click here for information regarding the specific requirements for shipment to your location
Seeds of Arabidopsis thaliana
Human hexokinase
brain/CNS neuroblastoma cell line
lung, small cell carcinoma
leukocyte
Homogenization
Mechanicaly: grinding, mixing, cutting, mortar with sand
Separation of cells
• centrifugation (6000 g, 10 min, 4°C) • filtration
Distribution of proteins
• Intracellular • periplasmatic • Extracellular
cytoplasmatic
periplasmatic
extracellular
cell
• Membrane proteins
Cell lysis
Methods • physical • chemical • enzymatic
Physical methods of lysis
• Mortar + glass beads, silica sand. • Alternating freezing and thawing • X-press (freezing and thawing)
X-press
piston
slit
• Douncer´s homogenizator
• Mini-BeatBeater – balotins – various dimensions
•French press - pass liquid suspension through small hole under high pressure (136 MPa)
• One Shot Disruptor
•Ultrasound - intezity - over 50 W/cm2 – cavitation
Chemical methods of lysis • osmotic lysis (erythrocyte)-suspension in hypotonic condition • change the gas pressure - nitrogen
Wikipedia
•detergents - compounds that lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid
CMC – critical micelle concentration C – concentration of detergent
•http://www.science.co.il/Biomedical/detergent_selection_table.pdf
critical micelle concentration (CMC) - the concentration of surfactants above which micelles form and all additional surfactants added to the system go to micelles.
•Y-PER Reagent significantly disrupts yeast cell wall and plasma membrane. Cells of Saccharomyces cerevisiae stain DY150 after lysis with Y-PER Yeast Protein Extraction Reagent. Arrows indicate disruption of cell wall, resulting in cell lysis.
•These images show a Streptococcus pyogenes cell experiencing lysis after exposure to the enzyme PlyC. (Credit: Daniel Nelson/UMD)
detergents •nonionic
•ionic •anionic
•kationic
•amfoteric
Detergent CMC mM
MMW Da
dialysis aplication
ANIONIC
SDS(dodecylsulfát sodný) 8,3 288,4 denaturation of proteins, for DNA, PAGE
DOC(deoxycholát sodný) 1-4 416,6 solubilization of membrane protein
N-lauroylsarkosin 7 488 solubilization of membrane protein
CATIONIC
CTAB (hexadecyltrimethyl amonium bromide)
4-5 337 ??? solubilization of membrane protein, complex with DNA, remove of polysaccharides
NONIONIC
Triton X-100 [octylphenolpoly(ethylenglycolether)n]
0,2 647 n=10
solubilizace proteinů
Tween 20 [poly(oxyethylene)n sorbitan-monolaurate]
0,06 imunobloty, ELISA
AMFOTERIC
CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)
4 614,9 solubilizace membránových proteinů
• Lysozyme – from egg whites
hydrolyse of 1,4-β-glycosidicbond between NAG a NAM
Yeast •zymolyase, lyticase – β-1,3-glucanase activity
Enzymatic methods of lysis Bacteria
Plant cells •cellulase, pectinase
Inhibitors of protease
Membrane process - filtration
•defined pore size = "cut off" (mm, Da), gravity or inert gas
microporous anizotropic
Produkt použití polyvinylidenfluorid PVDF low biding of protein polyethersulfon PES fast flow, low biding of protein esters of cellulose MCE filtration of buffers and particles polycarbonates smooth surface nylon and fiber mesh broad chemical compactibility, range 3
- 10 pH glass and a silica fibers coars filtration, solution with a high
number of particles teflon hydrofobic, org. solvents, agresiv
solutions
PVDF
0,1 - 5 μm
esters of cellulose nylon
0,22 - 180 μm 0,025 - 8 μm 0,7 – 3 μm
glass and a silica fibers
characteristic of membranes •dividing range – NMWL, MWCO, NCO •working temperatures, pressures and pH •resistance to chemicals
Membrane: Durapore Membrane Filters - PVDF (Polyvinylidene fluoride) Features: Low Protein Binding Specifications:
Pore sizes: 0.1, 0.22, 0.45, 0.65, 5 μm
Color: White
Surface: Plain
Wettability: Hydrophilic, hydrophobic
Thickness: 125 μm
Sterilization: by autoclave (121°C at 1 bar), EO or gamma
Operating temperature: 85°C max.
Gravimetric extractables: <0.5%
Protein binding capacity: 4 μg/cm²
•separation of coloidic or unsoluble particles (microorganisms) •0,05-10 µm •low pressure
Microfiltration
N2 pojistný ventil
míchadlo
retentát
permeát
membrána
•separation of proteins, virus •fractionation of proteins •size of pores 3 nm - 0,1 μm •pressure > 10 bar (145 psi, 1 MPa)
Ultrafiltration
Použití •desalting of whey •concentration
Nanofiltration
•separation of melecules with low mol. weight •size of pores 1 nm - 10 nm ∼ 200 – 15000 g/mol
- diffusion of low molecular weight substances
Dialysis
•http://www.inmed.cz/index.php?page=princip_dialyzy
Principle of hemodialysis
dialysis of 100 ml 5 M NaCl agains 1 litr of water
Precipitation
Fractionation of biomacromolecules on the basis of differences in solubility
• denaturation x precipitation
Franz Hofmeister (1850-1922)
for anions: SO42- > F- > IO3
- > NO2- >Br- > NO3
- > I- > CNS-
for cations: Li+ > Na+ > K+ > NH4
+ > Rb+ > Cs+
salting in
salting out
Ammonium sulfate precipitation
• very water-soluble • cheap • stabilization effect on proteins
The initial and final concentration of ammonium sulfate, expressed as percent saturation, are on the vertical and horizontal scales, respectively. The point of intersection of two lines drawn from these points indicates the number of grams of salt to be added to each littler of solution at the initial concentration to lead to the final concentration required.
Solid ammonium sulfate (grams) to be add to the 1 liter of solution.
G = wsat · (c2 - c1)/(csat - (Vspec/1000 ·132,14 · csat · c2))
temperature Solubility (NH4)2SO4
0 °C 70,21 g/100 ml 20 °C 75,16 g/100 ml 40 °C 80,83 g/100 ml 60 °C 87,21 g/100 ml 80 °C 94,30 g/100 ml
•G the weight of Ammonium Sulfate to add in grams per liter •wsat is the number of grams/Liter in a saturated solution of Ammonium Sulfate •c2 the molarity you want to achieve •c1 the starting molarity •csat is the molarity of a saturated solution of Ammonium Sulfate •Vspec is the specific volume of Ammonium Sulfate, which is the amount of volume 1g will add to an aqueous solution, which is about 0.54 ml
http://www.encorbio.com/protocols/AM-SO4.htm
Example: After the lysis of the cells, you have acquired a 20 ml of the extract of the protein (concentration of 0.5 mg/ml). You have choosen the ammonium sulphate precipitation (Mr 132,14) as a first step of purification of hexokinase. You find that hexokinase precipitate in 50% of saturation. That's why you have decided to do a two-step precipitation (40%, 50%). How would you follow (specific analysis and procedure)?
If you want to prevent a local increase of concentration, you will add the solution of ammonium sulphate. Count how many ml of 5 M ammonium sulfate, you must add to the 40 ml of the extract to the resulting concentrations were 0, 8 M.
mlVVV
VcVc
6,7)04,0.(8,0.5
..
1
11
2211
=+=
=
Precipitationwith organic solvents
• water-miscible organic solvents • low temperature (< 0 °C)!!!
Precipitation with acetonem (< 1µg/ml) add 5 volume of child (– 20°C) aceton and vortex 30 min at -20 °C. centrifugaton 5 min, 6,000 – 10,000 g. discard the supernatant and let the pellet air dry resuspend the pellet in buffer
aceton, EtOH, isopropanol, MetOH, propanol, diethyleter
Precipitation with organic polymers and organic compound
• PEG 4000 – 6000 – water soluble polymer, can be combined with ammonium sulphate precipitation
• tichloracetic acid – nucleic acid > 20 baze, proteins - prepare of samples for SDS-electrophoresis
Heat precipitation Relies on the differential resistance of proteins to denaturation and precipitation by heat. Perfect for the isolation of heat stable proteins
0102030405060708090
100
teplota
%
Bílkovina 1Bílkovina 2
Increase the temperature so that contaminating proteins are denatured while the desired protein remains stable and soluble.
The influence of pH on precipitation •Many proteins are precipitated from the solution in the pH that corresponds to pI •It is good to know the pI of the protein (chromatofokusace, isoelektrická fokusace)
Imunoprecipitation •antigens may be precipitated using the corresponding antibodies
•antibodies can be precipitated using the corresponding antigens
•antigens may be precipitated using insoluble form of antibody