chapter 10 analysis of proteins n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Chapter 10: Analysis of proteins PowerPoint Presentation
Download Presentation
Chapter 10: Analysis of proteins

Loading in 2 Seconds...

play fullscreen
1 / 24

Chapter 10: Analysis of proteins - PowerPoint PPT Presentation


  • 89 Views
  • Uploaded on

Chapter 10: Analysis of proteins. Purification schemes: 1. soluble recombinant proteins 2. insoluble recombinant proteins that are produced as inclusion bodies in the cytoplasm of host cells. 3. soluble proteins present in the natural host cells

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Chapter 10: Analysis of proteins' - kiora


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide3

Purification schemes:

1. soluble recombinant proteins

2. insoluble recombinant proteins that are

produced as inclusion bodies in the

cytoplasm of host cells.

3. soluble proteins present in the natural host cells

4. membrane-associated and poorly soluble

proteins (non-recombinant).

slide9

Protein concentration determination methods:

1. A280 (UV lamp)

if A is > 2, dilute the sample.

2. Bradford method (Bio-rad)

Stain protein with Commassie blue solutions,

and read A595.

slide10

Quantitative amino acid analysis

A good way to check purity and confirmation of the protein sequence.

Protein sample is hydrolyzed by 6M HCl,

then Beckman 6300 system for ninhydrin detection.

Frequently amino acid residues in a protein: Gly, Ala, Ser,

and Leu.

Rare amino acid residues in a protein: Trp, Met, His, Cys,

and Arg.

Calculate ratio of each aa (Ratio1)

Ser, and Thr (loss rate is high in the method) and rare aa (His,

and Met) are taken out from the total amounts,

re-calculate the ratio (Ratio 2).

slide14

Safety elctrophoresis operation:

To start:

Power turn to zero

Turn on power

Hook gel apparatus (use one hand with one outlet at a time, the other hand is free. )

Set to fixed current, but sometimes fixed voltages, and run .

To stop:

Power turn to zero

Power off

Unhook gel apparatus (use one hand with one outlet at a time, the other hand is free.)

DO NOT unhook gel apparatus first, before turn the power off.

Otherwise, electrical charges stays in power even the power is off and will cause shock.

slide16

Polyacrylamide gel elctrophoresis (PAGE)

Laemmli method: two buffer systems, discontinuous

Denature PAGE: SDS-PAGE

Sample proteins are solubilized by boiling in the presence of

SDS.

2-Me and DTT are added during solubilization to reduce

disulfide bonds.

SDS (detergent with polar head and non-polar tail) for coat

protein with negative charge.

Protein is denatured.

- Negative charge density is same with different proteins.

- Negative charges are proportional to the MW of the proteins.

slide17

Denature PAGE: SDS-PAGE

1. Tris –glycine buffer for separation of MW > 14 kd.

(because SDS move to the 14 kd and below area)

2. Nonurea peptide separations with Tris buffer.

Increased buffer concentrations for separations to 5 kd.

3. Tris – Tricine buffer.

for separation of MW of 10-15 kd

slide18

Non- Denature PAGE

No boiling, SDS, 2-ME or DTT, others are all same.

(Some proteins will move to anode and into buffer, the pH may need to be changed.)

slide19

2-D gel electrophoresis

1-D: isoelectric focusing

use Bio-Rad Tube Cell 175, or tube cell adaptors

with mini-protean III.

Prepare Gel with ampholytes, will generate pH gradients

when electrophoresed.

Proteins will stay at pH position equivalent to its PI.

commercial strips are available.

NEPHGE (non-equilibrium pH gradient electrophoresis)

Modifications can resolve very basic or acid proteins.

2-D: SDS-PAFE

slide20

Staining proteins in gel

1. conmmassie blue staining

non-specific binding to proteins,

detection limit: 0.3 to 1 μg/ protein band.

2. silver staining (S-H group, and C=O group)

some proteins cannot be stained.

detection limit: 2 to 5 ng/ protein band

nonammoniacal silver staining

detect proteins that cannot proteins by 2.

3. Fluorescent staining

SYPRO red, ruby or orange

See under 300nm UV illuminator, or a laser scanner.

Staining procedures for IEF, native gel and SDS-PAGE might be different.

slide21

Immunoblotting

Tank transfer

Semi-dry

(a protocol for blotting of a CB-stained gel is provided)

Ponceau S can reversible stain the membrane.

CB stains permanently, cannot do further hybridization.

slide22

Immunodetection

1. directly conjugated secondary antibody (eg. AP or HRP-conjugated)

2. Avidin-biotin coupling to secondary antibody

Visualization

with chromogenic substrates

HRP- or Ap-based.

with luminescent substrates

HRP- or AP based.