PROTEIN PURIFICATION AND ANALYSIS - PowerPoint PPT Presentation

Loading...

PPT – PROTEIN PURIFICATION AND ANALYSIS PowerPoint presentation | free to download - id: 4b8632-NDUyY



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

PROTEIN PURIFICATION AND ANALYSIS

Description:

PROTEIN PURIFICATION AND ANALYSIS Assays Need measures for the object (enzyme activity, chromophore, etc.) and for total protein concentration: Use spectrophotometry ... – PowerPoint PPT presentation

Number of Views:854
Avg rating:3.0/5.0
Slides: 16
Provided by: Terence50
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: PROTEIN PURIFICATION AND ANALYSIS


1
PROTEIN PURIFICATION AND ANALYSIS
2
(No Transcript)
3
(No Transcript)
4
Assays Need measures for the object (enzyme
activity, chromophore, etc.) and for total
protein concentration
5
(No Transcript)
6
  • Use spectrophotometry for
  • Measuring substrate or product concentration if
    one is
  • colored (absorbs UV or visible light).
  • 2. Measuring substrate or product concentration
    if one reacts
  • with something to form a colored compound.
  • 3. Measuring the concentration of total protein
    (by UV
  • absorbance A280 1 _at_ 1 mg/ml) or of a specific
    protein (like
  • hemoglobin) that contains a colored
    chromophore.
  • Measuring the concentration of protein by
    reacting it with
  • a reagent to form a colored compound. (E.g.,
    cys, tyr, trp
  • reduce Cu2 to Cu, which forms an intense blue
    with BCA
  • Coomassie reagent turns blue in hydrophobic
    environment.)

7
Measuring purification Specific activity (SA)
total enzyme activity/total protein or activity
concentration/protein concentration 1 IU
(international unit) 1 µmol substrate used
(product formed)/min-mg protein Purification
at step x SA of step x / SA of crude
extract One measure of purity is constant
SA. Note if the protein of interest is 0.2 of
the total, then you require 500-fold
purification for purity if each step gives
3-fold purification, then you need 5-6 steps if
each step causes some loss of the protein of
interest (e.g. 50), you need to start with
30-60-times more enzyme than you will recover.
8
Purification techniques size, charge, specific
binding
Size exclusion chromatography
9
(No Transcript)
10
(No Transcript)
11
(No Transcript)
12
Two-dimensional gel electrophoresis IEF
followed by SDS-PAGE
13
(No Transcript)
14
Affinity chromatography separate by biological
specificity 1. Attach ligand to insoluble
column material 2. Combine protein mixture with
column enzyme binds to ligand 3. Wash column
to remove unwanted material 4. Elute enzyme with
substrate or change in pH (to reduce binding)
15
Summary Enzyme purification involves breaking
cells and stabilizing pH, proteolysis, measuring
enzyme activity and protein concentration, a
series of separation techniques Spectrophotometry
is useful for measuring protein concentration
and, often, enzyme activity Exclusion
chromatography, electrophoresis, isoelectric
focusing, and affinity chromatograph are useful
methods for purifying proteins
About PowerShow.com