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Biochemistry Protocols
Amino Acids and Proteins
Protein Purification
Protein Crystallization
Proteolytic Cleavage
Peptide Mapping
Lipids
Lipid Purification
DGK Assay
Liposome Swelling Assay
Lipid Extraction
Seperation and Quantification
Lipid Transmethylation
Enzymes
ATPase Assays
Kinase and Phosphotase Assays
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To characterize the structure, function and the interactions of a protein, isolating a single protein from a complex slurry of biological culture is known as protein purification. There are multiple steps and ways to isolate a single protein from a cellular matrix. These separation processes exploit protein size, binding and physico-chemical properties such as pH.
Small Scale GST-Tag Purification Under Nature Conditions
Denaturing purification of insoluble bacterial GST-fusion Proteins
GST Fusion Protein Prep (from bacteria) with beads and thrombin cleavage
GST Fusion Protein Purification from Yeast
To determine the structure of a protein, crystallographic analysis by X-Ray diffraction or Nuclear Magnetic Resonance can be carried out. A protein is a macromolecule with many degrees of freedom; as such, these molecules (along with RNA) must keep in tact the molecule's tertiary structure and folding for proper crystallization since it is the tertiary folding that maintains structural stability.
Protein crystals are grown in solution and induced to nucleate based on solution conditions which favor the growth of a large crystal (for the sake of resolution) and then will "salt-in" to favor multiplicative crystal growth, so as to seed one very large crystal per solution. This process is known as the hanging drop method.
Hanging Drop/High-Throughput Basics
Hanging Drop Crystallization
High-Throughput Crysallization
High-Throughput Protein Crystallization, Practical Course (EMBL-Hamburg)
X-ray Crystallography in 5401 Seconds (Manfred Weiss)
Peptide bonds between amino acids in proteins can be broken by a process known as proteolytic cleavage. This is carried out by lytic enzymes such as peptidases, proteases or proteolytic cleavage enzymes.
Naturally, proteins undergo proteolytic processing as a form of structural integrity conservation. This may be to counter misfolding due to environmental conditions or other functional mechanisms such as correcting structure in protein secretion or membrane implantation. Taking advantage of this mechanism, it is possible to to work with specific peptide fragements rather than entire proteins.
Partial Proteolytic Cleavage
In-Situ Proteolysis
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Peptide mapping involves controlled cleavage of a pure protein with small amounts of a pure protease to generate peptides of characteristic, reproducible sizes. These peptides can be separated on PAGE to produce a â€fingerprint†characteristic of the protein. Peptide mapping can map cleavage sites in an unknown protein, or it can identify an unknown protein based upon its fingerprint identity with a previously tested sample.
The polyacrylamide gel used can be either denaturing or non-denaturing, but SDS PAGE is most often used because it gives molecular weight information about the peptides produced. Small amounts of protease are used, so that minor variations in time and temperature of incubations will not overly perturb the results.
Proteins for peptide mapping can be taken from bands sliced out of electrophoresis gels, or purified by standard means. Protocols are provided for the mapping of a pure protein, a protein in an acrylamide gel, and a protein isolated from National Diagnostics' ProtoPrep II Matrix.
Tryptic or Chymotryptic Peptide Mapping
Cynaogen Bromide Peptide Mapping
The Cleveland Method: SDS-PAGE Peptide Mapping
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Column Purification of Demethylated Sphingomyelin
LIPID EXTRACTION
Papers: Membrane Lipids :: isolation & purification
A Method for Micro-Scale Isolation and Purification of Gangliosides
Purification of fatty acids before analysis
DGK Membrane Preparation
Liposome Preparation
Notes
Thin Film Hydration
Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery
WARNING!
(particularly to students):
Please Remember VERY WELL that while Green Chemistry techniques and reactions may utilize less toxic chemicals, often times the by-products that are produced are more dangerous and environmentally damaging than direct reaction techniques.
Be sure to thoroughly READ AND WORK OUT (with proper MSDS files) all reactions for all potential hazards.
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