Prepared by: Nor Esnita Mohd Salleh
Amino acids which are linked by peptide bonds between amino group and carboxyl group
Enzymes, hormones, receptors, antibody, Enzymes, hormones, receptors, antibody,
molecule carriers and muscles.
Express the genetic traits in each organism.
Cell disruption
Differential centrifugation Differential centrifugation Contaminants removal
Protein assay
Protein separation and purification
Physical methods Physical methods Physical methods
Physical methods Chemical methods Chemical methods Chemical methods Chemical methods
Grinding:
With liquid nitrogen in mortar to get the cell in powder form.
Blend using high-speed blender.
Homogenizer such as French press
Squirting the cells at the high pressure through the small orifice.
Sonicator
Using ultrasonic vibration
Osmotic lysis
Suspended cells in hypotonic solution.
Enzyme
Lysozyme: hydrolyse the cell wall.
Lysozyme: hydrolyse the cell wall.
Detergent or organic solvent
Sodium dodecyl sulphate (SDS),Triton X-100
Alkali solution
Sodium hydroxide (NaOH)
Pellet (nuclei I)
Lysate cells
Supernatant (cyto I)
+ STMDPS buffer, homogenize, centrifuge 800g, 15
minutes
Supernatant
(cyto II) Pellet (nuclei II)
Centrifuge 800g, 15
minutes
+ STMDPS buffer, homogenize, centrifuge 80 000g, 35 minutes and, filter
Pellet Supernatant
Nuclei Protein
Resuspend in NE buffer
Centrifuge 9 000g, 30
minutes
Resuspend in NET buffer, centrifuge 9 000g, 30 minutes
Supernatant
Pellet Supernatant
Soluble proteins
Nuclear membrane protein
Other proteins that tightly bound to DNA
Supernatant (discard)
Supernatant (cyto II) Supernatant (cyto-I)
Centrifuge 6000g, 15
minutes
Supernatant (cyto III)
Supernatant (cyto III) Pellet
Pellet
Resuspend STMDPS buffer, centrifuge 6000g,
15 minutes
Supernatant (discard)
Pellet (mitochondria)
Resuspend HDP Centrifuge
6000g, 15 minutes
(discard) Resuspend HDP
buffer, incubate, sonicate and centrifuge 9000g,
30 minutes
Mitochonrdria matrix protein
Mitochondrial membrane protein
Supernatant Pellet
Resuspend in ME buffer, centrifuge 9000g, 30 minutes
Supernatant
Mitochondria protein
Cyto III
Supernatant (cytosol)
Centrifuge 100 000g, 1h
Pellet
Cytosol protein
Resuspend in ME buffer, incubate for 1h, centrifuge 9000g,
30 minutes
Supernatant Microsome
protein
Microsome and cytosol protein
Phospholipids
Detergent such as Triton X-100,CHAPS (zwitterionic detergent) and Nonidet NP-40 which able to increase the solubility of hydrophobic proteins.
Nucleic acid
DNAse and RNAse
Sonication where this technique can break the nucleic acid into little fragments.
Precipitation using protamine sulphate or
polyethyleneimine
Sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE)
Separate the proteins based on molecular weight.
Separate the proteins based on molecular weight.
SDS is applied to coat the protein molecules with
negatively charge then allowed the protein to migrate towards anode in the
electrophoresis buffer.
SDS also lead to denature protein and lead to the
primary structure of the protein.
Spectrophotometry
Using spectrophotometer to get the
absorbance of the proteins at 280nm of wavelength.
Compare the absorbance of the protein
Compare the absorbance of the protein with the absorbance of bovine serum
albumine (BSA) to get the quantity of the
protein (µg).
STMDPS buffer STMDPS buffer STMDPS buffer STMDPS buffer
Initial homogenization buffer.
Containing:
Reagent Function
Dithiothreitol (DTT) Reducing agents; to prevent the oxidation process.
Spermine Alkaline conditions for protein,
maximize protein extraction,
precipitate nucleic acid and keep protease activity low.
Spermidine Precipitate of DNA
NE buffer NE buffer NE buffer NE buffer
Nuclear protein extraction buffer.
Containing:
Reagent Function
N-2-hydroxyethylpiperazine- N’-2-ethanane-sulfonic acid (HEPES)
pH stabilization
Ethylenediaminetetraacetic acid (EDTA)
Chelating agent for protease inhibitors activity.
Glycerol Stabilizing agent.
MgCl2 Metal ion for protease inhibitor
activity
NET buffer NET buffer NET buffer NET buffer
Nuclear protein extraction buffer.
Containing
- Triton-X-100
- Dithiothreitol (DTT)
- Phenylmethyl-sulfonyl fluoride (PMSF) - Phenylmethyl-sulfonyl fluoride (PMSF)
Reagent Function
Dithiothreitol (DTT) Reducing agents; to prevent the oxidation process.
Triton-X-100 Detergent; increase the solubility of hydrophobic protein.
Phenylmethyl-sulfonyl fluoride (PMSF)
Protease inhibitor.
HDP buffer HDP buffer HDP buffer HDP buffer
Hypotonic lysis buffer to extract soluble mitochondrial protein.
Containing:
Reagent Function
Reagent Function
Dithiothreitol (DTT) Reducing agents; to prevent the oxidation process.
Triton-X-100 Detergent; increase the solubility of hydrophobic protein.
Phenylmethyl-sulfonyl fluoride (PMSF)
Protease inhibitor.
N-2-hydroxyethylpiperazine -N’-2-ethanane-sulfonic
acid (HEPES)
pH stabilization
ME buffer ME buffer ME buffer ME buffer
Extraction buffer for mitochondria and microsome membrane.
Containing:
Reagent Function
Reagent Function
Dithiothreitol (DTT) Reducing agents; to prevent the oxidation process.
Tris-HCl Alkaline conditions for protein, maximize protein extraction,
precipitate nucleic acid and keep protease activity low.
N-2-hydroxyethylpiperazine- N’-2-ethanane-sulfonic acid (HEPES)
pH stabilization
Glycerol Stabilizing agent.
Triton-X-100 Detergent; increase the solubility of hydrophobic protein.
Phenylmethyl-sulfonyl fluoride (PMSF)
Protease inhibitor.
Protein extraction process must be done immediately after sample collection to prevent protein degradation.
Cell disruption must be done in cold temperature as cold as possible. Maximum temperature that suggested is
as possible. Maximum temperature that suggested is 4 ° C.
Minimize the sample treatments to avoid protein losses.
pH that suitable for protein extraction is 7.0 to 8.5.
1. Aususel, F.M., Moore, D.D., Struhl, K., Brent, R.,
Scidman, J.G., Smith, J.A. and Kingston, R.E., 2002.
Short Protocol In Molecular Biology. Fifth Edition. USA:
John Wiley & Sons.
2. Cox, B., and Emili, A., 2006. Tissue subcellular
fractionation and protein extraction for use in mass- fractionation and protein extraction for use in mass-
spectrometry-based proteomics. Nature Protocols. Vol.
I. Nature Publishing.
3. Simpson, R.J., 2003. Proteins And Proteomics A Laboratory Manual. USA: Cold Spring Harbor Laboratory Press.
4. Westermeier, R., and Naven,T., 2002. Proteomic In
Practice. Germany: Wiley-VCH.
