Protein purification:

Concentration determination

Lani Keller
Bio 606
9/19/2013
First we are going to review of what
we have done the last two labs
 Bacterial Cell Disruption
Cell Disruption (Lysis) Buffer
1. Buffer (PBS)
6-HIS-GBP 2. Salt (NaCl)
Construct 3. Protease Inhibitors (PMSF + tablets)

4. Lysozyme
5. Imidazole (for later purification steps)

Supernatant-
soluble
Fraction
“Cleared Lysate”
Pellet-
Sonication Centrifugation
insoluble
material
Whole Cell Lysate & debris
 Ni-Affinity Chromatography
Supernatant-
soluble
Fraction
“Cleared Lysate” Wash 3X to
replace ethanol
with wash buffer

1. Load Column 2. Wash Column 3. Elute Column

Batch Binding
Beads +
“cleared lysate”

Keep Elutions!
Discard Flow-through
 Predicted 6-His-GFP Ni-NTA Elution Chromatograph
100

90

80

70
Amount of 6-His-GBP

60

50

40

30

20

10

0
1 2 3 4 5 6 7 8 9 10 11

Fraction (Elution) Number

 Learning Objectives

• Be able to explain the importance of determining

protein concentration

• List three methods for determining protein

concentration

• Describe the physical basis, advantages, and

disadvantages for each method

• Construct a standard curve and determine the protein

concentration of an unknown sample
Determining Protein Concentration…Why?
1. Figure out where your protein is?
2. Which steps to use next depends on how much
protein you have?
3. To study your protein you need to know how much
4. Diagnostics
– Determine disease, test diseases, vaccines
– 33.3 billion dollars last year for the sale of just 10 protein
therapeutics (including insulin, growth hormone)
– All protein therapy started by studying purified proteins
Three Methods for Determining Protein
Concentration

1. Absorbance measurements of 280nm (A280)

Quick and dirty

2. Lowry and BCA (bicinchoninic acid) assay

Copper based methods

3. Bradford assay
Most commonly used in labs
1. Absorbance measurements (A280)
Physical basis: Several amino acids (primarily Tryptophan
and Tyrosine) absorb ultraviolet light at 280 nm.

Advantages:
1. Quick, Direct
2. Doesn’t “use” sample

Disadvantages:
1. Not strictly quantitative- protein has lots/few aromatic amino acids
2. Relatively insensitive (0.2 to 2.0 mg/mL)
3. Interference issues (nucleotides)
2. Lowry and BCA (bicinchoninic acid) assay

Physical basis: Reduction of Cu2+ to Cu1+ by amides.

Based on the reaction of copper ions

with the peptide bonds

Advantages:
1. Highly sensitive (5-100 µg/mL)
2. Little variation among proteins
Disadvantages:
1. Interference issues (detergents, reducing agents, buffers)
2. Relatively slow (~40 min)
3. Many solutions required (CuSO4, Na3C6H5O7, Na2CO3, NaOH,
Folin-Ciocalteu phenol reagent)
4. Proteins are irreversibly denatured
 3. Bradford assay
Physical basis: Coomassie Brilliant Blue G-250 changes color upon
binding protein.

Advantages:
1. Highly sensitive (25-200 µg/mL) so you use little protein
2. Relatively fast (~10 minutes)

Disadvantages:
1. Interference issues (detergents)
2. Proteins become irreversibly denatured
 Biorad protein assay (Bradford)

Coomassie Brilliant Coomassie Brilliant Blue G-

Blue G-250 alone 250 + Protein

(Absorbs at 465) (Absorbs at 595)

 Generation of a Standard Curve

BSA: Bovine Serum Albumin

Abs595
Generate a standard
curve with solutions
that have a KNOWN
concentration.

Concentration (g/mL)
 Spectrophotometers
• Incident Light at 595 nm
• Protein-Dye Complex in Cuvette Absorbs Light
• Detector Detects Transmitted Light - Subtraction Obtains
Absorption
• Absorbance vs. Wavelength
– Sensitivity – Smallest Sample Size That an Assay Can
Detect

From the Chemistry Hypermedia Project, http://www.chem.vt.edu/chem-ed/index.html

Calculating Protein Concentrations from a
Standard Curve

STEP 1: Determine the protein concentration you put into the assay

Use the Linear Equation y = mx + b

m = Slope
b = y-intercept

STEP 2: Calculate the protein concentration in the original fraction

Use the equation C1V1 = C2V2

C1 = Protein concentration of the elution fraction (mg/ml)

V1 = Volume of test dilution added to assay (ie. 50ul, which is 0.050ml)
C2 = Protein concentration found using linear equation above (x).
V2 = Total Volume of Assay (1.5 ml)

**Convert your units appropriately**

 This week’s lab warnings!
Do NOT put your entire sample into Bradford assay!

This lab will be the longest lab yet, may take longer than 4h.

There is only one nanospec and five groups!! Every group will
need to generate a standard curve and determine the
concentration of their proteins.