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
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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
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.
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,
Rare amino acid residues in a protein: Trp, Met, His, Cys,
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).
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 .
Power turn to zero
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.
Laemmli method: two buffer systems, discontinuous
Denature PAGE: SDS-PAGE
Sample proteins are solubilized by boiling in the presence of
2-Me and DTT are added during solubilization to reduce
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.
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
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.)
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
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.
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.
(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.
1. directly conjugated secondary antibody (eg. AP or HRP-conjugated)
2. Avidin-biotin coupling to secondary antibody
with chromogenic substrates
HRP- or Ap-based.
with luminescent substrates
HRP- or AP based.