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 1 st term of exam
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Isolation and Analysis of Biomacromoleculesold-biomikro.vscht.cz/vyuka/ibe/1_lecture_2015.pdfsalting in salting out Ammonium sulfate precipitation • very water-soluble • cheap
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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)
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
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
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
•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
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.
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
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