Methods of Protein Purification

Methods of Protein Purification. Rachel Britt Cox Lab Biochemistry Addition, Rm 337 britt@wisc.edu Biochemistry 660 September 9, 2009. Why purify a protein?. Characterize function, activity, structure Use in assays Raise antibodies many other reasons ... . Why purify E. coli DinI?.

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protein purification

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Protein Purification

Resources. Protein purification: A practical approach. (Harris

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Protein Purification Processes

Bioseparation Engineering. Protein Purification Processes. Protein Purification Processes. Final product requirement – define (99% ~ 99.999%). Process design approaches. Follow examples Modify the process (new concept can be introduced).

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Protein Purification and Analysis

Protein Purification and Analysis. Numbers of genes: Humans ~40,000 genes Yeast ~6000 genes Bacteria ~3000 genes Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Some proteins expressed at high levels (collagen, hemoglobin)

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Protein Purification

Protein Purification. Molecular weight Charge Solubility Affinity. Molecular Weight. Ultracentrifugation Dialysis Gel filtration SDS PAGE. Molecular Weight. The lab in week 6 and 7 will involve separating a protein mixture by molecular weight using 2 methods: gel filtration and SDS PAGE

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Protein Purification and Characterization

Protein Purification and Characterization. Why Study proteins? IMPORTANT FACTORS IN PROTEIN PURIFICATION Starting materials tissues, cells or clones expressed in E. Coli or animal cells Decisions- quantity of protein, protein modification availability of samples, is it cloned yet, expense.

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Protein Purification Strategies

Protein Purification Strategies. Course: Methods in protein chemistry Rahman M. Mahfuz ur 2012/01/11 SLU. Objectives. To seperate a particular protin from all other proteins and cell components There are many types of proteins within an ogranism

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Protein Purification

Protein Purification. Tutorial Minnie Murugesan Dr. Scott’s Group 07-12-05. Eventful Protein Purification. Grow cells in media (vector+tag) Centrifuge, Collect the pellet Lyse the cells (appropriate buffer). Pilot Expression SDS PAGE, Assay. Purification Strategy. Characterization

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Protein Purification

Protein Purification. Why purify Proteins?. Characterize Function Activity Structure Study protein regulation and protein interactions Use in assays Produce Antibodies Perform structural analysis by X-Ray and Crystallography. Where do you get proteins?. Nature Proteins (from organism)

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Protein Purification and Expression

Protein Purification and Expression. MCB 130L, Lecture 2. Review of DNA Lab. PCR Restriction Digests Agarose Gel Electrophoresis. Central Dogma of Molecular Biology. Proposed by Francis Crick, 1958. Why purify a protein?. To study its function To analyze its physical properties

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GFP protein purification lab

GFP protein purification lab. What are we trying to do. 7.3- Use E.coli that contains pGLO plasmid w/ GFP gene to express GFP protein. 2ml of culture. What are we trying to do. 7.3- Use Lysozyme enzyme b reak open bacterial cells to release GFP protein ( TTh )

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Protein Purification

Protein Purification. Contains protein and non protein. salt. Non protein. Salt + protein. Add buffer. Buffer Solution contains: Mixture of salt and protein. Reduce the salt concentration (by dialysis). Dialysis: Reducing the salt concentration in the mixture solution. gf.

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Protein purification

Protein purification. Chroma tography – Mikhail Tsvett (1901) pioneered the technique while attempting to separate yellow and red pigments from green leaves.

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Protein Purification & Crystallization

Protein Purification & Crystallization. From cDNA to crystal structure. Design of the (best) constructs. BLAST-P your sequence (set PDB as database), find homologues; identify domains

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Protein Production and Purification

Protein Production and Purification. “Why the medium composition counts.". The E. coli System. Variables in Protein Production. Gene. Vector. Host. Production. Codon Usage mRNA Structure GC Content Regulatory Motifs Repeats. Replication Origin Promoters Ribosome Binding

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Protein Purification

Protein Purification. Protein Purification. What do you know about proteins? Why do we need to purify proteins? What are you curious about?. Bacterial chromosome. What is Transformation?. Uptake of foreign DNA, often a circular plasmid. Plasmid.

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Protein Purification

Protein Purification. Initial Questions How much and how pure? application source feasibility Native configuration? functional/structural (yes) sequence (no) antibody (maybe) Detection method? functional assay band on gel. Why purify proteins? functional and/or structural studies

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Lecture 8: Protein purification

Lecture 8: Protein purification. Protein purification. Column chromatography. After the initial fractionation steps we move to column chromatography . The mixture of substances (proteins) to be fractionated is dissolved in a liquid or gaseous fluid called the mobile phase .

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Protein Purification

Protein Purification. Fig. 5-CO, p.113. Learning Objectives. Protin Purificartion & Characterizing Techniques. 1. How Do We Extract Pure Proteins from Cells?

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Protein Purification

Protein Purification. Fig. 5-CO, p.113. Protin Purificartion & Characterizing Techniques. 1. How Do We Extract Pure Proteins from Cells?

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