Lecture 6 Flashcards
What does ProParam give?
obtain molecular mass, pI, number of each type of residue
What does Blast give?
obtain related protein sequences.
What is the sequence format computers use?
FASTA
Ways to obtain a protein sample in molecular biology?
- Clone the gene from genomic DNA or cDNA or synthesize the gene
- Over express protein in bacterial or other host
- In vitro expression
(need: plasmid with cloned gene, RNA polymerase, NTPs, ribosomes, amino acids)
Ways to obtain a protein sample in chemistry?
- Chemical synthesis.
- Has the advantage of not being restricted to the standard 20 amino acids.
- There is currently a length limit.
Examples of proteins purified from natural sources?
Digestive enzymes (pepsin, chymotrypsin, trypsin)
α-keratin (Wool)
Collagen (gelatin)
Serum albumin (bovine serum albumin or BSA)
Hemoglobin from RBCs (Horse)
Myoglobin from muscle (whale)
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), first major step of carbon fixation – CO2 glucose (most abundant protein on earth)
Lysozyme
Ribonuclease A
Photosynthetic rxn center (photosynthetic bacteria)
Eqn to determine possible potential polymers?
N^L
N = # of diff monomers
L = length of polymer (monomer units)
How many possible potential polymers are there for dipeptide Ala + Gly?
N^L
2^2
= 4
How to optimize rxn so polypeptide synthesis?
- Protection group e.g. Fmoc
- Prevents it from activating as a nucleophile - Leaving group e.g. DCCD
- Inc electriphicity - Polystyrene bead support
What is the direction of synthesis of polypeptides?
C -> N
What are the 4 general steps in preparing pure, monodispersed protein
- Fractionation
- Chromatography
- Concentration
- Analyzing
3 examples of 3 fractionations?
- Ammonium sulfate cuts (salting-out, precipitations, Hofmeister series)
- Organic extraction
- Temperature
3 examples of chromatography?
- Ion exchange – separation by charge (anion exchange, cation exchange)
- Affinity
- Gel-filtration (size-exclusion)
2 examples of concentrating?
- AS precipitation, solubilize in smaller volume.
2. Ultra-filtration
5 examples of analyzing techniques?
- Concentration determination – BCA (Cu reduction), Bradford assay (Coomassie G-250), based on sequence calculate theoretical, use Beers eqn.
- SDS-PAGE
- N-terminal sequencing
- Mass-spectrometry
- Analytical gel-filtration / light scattering
Draw protein solubulity vs. ssalt conc
N/A
What are the 3 maintypes of ion exchange?
- Ion exchange
- Affinity/ HIC
- Size exclusion
Two types of ion exchange chromatography?
- Proteins with a net negative charge (pI below 7) will likely stick to an anion-exchange resin
Example: Q-sepharose
0 Proteins with a net positive charge (pI above 7) will likely stick to an cation-exchange resin
Example: S-sepharose
What is affinity chromatography, using His?
Engineer 6 His tag at N terminus to purify protein out of all other proteins in lysate
- waters replaced by the imidazole side chains of 2 histidine reisues in the 6His-tag. The protein is usually eluted off using imidazole to complete off His-tag
What are the 2 protein fusion affinity tags?
- Maltose Binding Protein (MBP)
- Amylose column - Glutathion S-transferase (GST)
- Glutathione column
Fxn of dialysis?
For changing buffers or desalting.
The tubing comes in different “Molecular Weight Cut Off” (MWCO).
A good type of tubing is Spectra/POR.
Can also use gel-filtration / desalting column: Sephadex G-10
What is SDS Page?
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
What is native blue electrophoresis?
Instead of SDS the anionic dye Coomassie Brilliant blue R250 is used for solubilization of proteins, in the absence of reducing agents.
What is isoelectric focusing for?
Experimentally determines the isoelectric point (pI)
Steps in isoelectric focusing?
- Cast gel
- Establish gradient
- Add sample
- Run gel
What is 2-D electrophoresis?
~ 10 000 different proteins can be distinguished
3 ways to concentrate protein?
- Precipitation
- Compressed gas ultra-filtration
- Centrifugation ultra-filtration
How can you use precipitation to concentration proteins?
You can use precipitation to concentrate proteins. You use concentrated ammonium sulfate to precipitate the protein and then you can resuspend the protein precipitate in a smaller volume of buffer. This sometimes does not work.
How can you use compressed gas ultra filtration to concentration proteins?
Compressed gas is used to move liquid through a membrane with a molecular weight cut off (example, molecules smaller than 10 KiloDaltons (including water) will flow through the membrane, and the protein will concentrate. This device is called the Amicon Stir-Cell.
How can you use centrifugation ultra-filtration to concentration proteins?
replace gas pressure with centrifugal force
How to determine protein concentration by UV spec?
- Calculate theoretical extinction coefficient based on the amino acid sequence via the program ProtParam
- Measure absorbance (UV-vis spectrometer) at 280nm, Tryptophan contributes the most to the absorbance at this wavelength
What is the Beer-Lambert equation?
A / Eb = c
A = absorbance (unitless) E = extinction coefficient b = path length (1cm) c = concentration (m)
How to determine protein concentration by chemical methods?
- The Bradford protein assay (Coomassie Dye-based Protein Assays)
- Absorbance shift (465 nm to 595 nm) upon binding to protein
- BCA protein assays (BCA = bicinchoninic acid)
562 nm, reduction of Copper, binding of BCA
Issues with bradford protein assay?
- Depend on a standard curve using BSA (bovine serum albumin) which is of course not the protein you are interested in.
- There are potential effects from the other molecules in solution, buffers, detergents, etc..
3 ways to access purity of protein sample?
- Specific activity (activity/total protein) can be used to assess protein purity
- Mass spectrometry
- Electrophoresis
- Coomassie Brilliant Blue (R-250) ~ 100 ng
- Silver staining ~ 10 ng
How to store proteins?
- Avoid freeze/thaw – aliquot into usable volumes, eg. 250 μL.
- Sometimes it is helpful to add compounds like glycerol to prevent protein damage that can occur during freezing.
- Quick freezing in liquid nitrogen is sometimes used to help prevent problems with freezing.
How is purifying membrane proteins different from soluble proteins?
Purified the same way as soluble proteins, but you first need to extract them from the membrane and you need to always have detergents/lipids present.
What is the aggregation #?
of detergent molecules in the micelle
