Beer-Lambert Law

Beer-Lambert Law: I0 = intensity of incident light I = intensity of transmitted light log(I /I) = ebc e = molar absoptivity coefficient in cm2 0 mol-1

-1 e = A/cb c = concentration in mol L b = pathlength of absorbing A = ebc solution in cm-1

A = absorbance = log(Io/I) A = ec (when b is 1 cm)

ℓ

0.1 cm http://www.hellma-worldwide.de/en/default.asp For example…

• Protein abs @ 280 (1g/L) = 1.0 • Abs is 0.67 • Thus – Abs/abs(1g/L)=0.67mg/ml Protein purification and characterization

Exercise #5 Affinity Chromatography Protein Purification & Characterization What is Chromatography?

Invented by a Russian botanist named Mikhail Tswett in 1905. He separated plant pigments using glass columns packed with calcium carbonate. Before we proceed…

How we conduct chromatography?

FPLC (AKTA Purifier) Gravity-assisted glass column עקרונות ההפרדה Affinity Chromatography

Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatography matrix.

•What are the advantages of this technique? •What’s the importance of the reversible binding? Customization of affinity column

• Choosing the right ligand (verify dissociation) • Choose the right matrix: – Hydrophilic

– Large spaces Aldehyde – Rigid activation – Inert – Chemically stable • Attaching ligand to matrix

For example, immobilization of IgG to agarose beads Choice of ligand ? • Enzyme ↔ substrate analogue, inhibitor, cofactor. • Antibody ↔ antigen, virus, cell. • Lectin ↔ polysaccharide, glycoprotein, cell surface receptor, cell. • Nucleic acid ↔ complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein. • Hormone, vitamin ↔ receptor, carrier protein.

The dissociation constant (kD) for the ligand - target complex should ideally be in the range 10-4 (enzyme and a weak inhibitor) to 10-8 M (hormone and hormone receptor) in free solution. Choice of a Matrix • Hydrophilic – to avoid non-specific interactions • Large spaces – to increase surface area and enable large protein to enter • Rigidity – to withstand the pressure of the solvent • Inert – so it won’t affect the binding between the protein and the ligand • Chemical stability

Methods for protein’s elution

Method 1: The simplest case. A change of buffer composition elutes the bound substance without harming either it or the ligand.

pH elution A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. This change may affect the binding sites directly, reducing their affinity, or cause indirect changes in affinity by alterations in conformation. Ionic strength elution The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a linear gradient or in steps. Methods for protein’s elution Method 2: Extremes of pH or high concentrations of chaotropic agents are required for elution, but these may cause permanent or temporary damage.

Chaotropic eluents If other elution methods fail, deforming buffers, which alter the structure of proteins, can be used, e.g. chaotropic agents such as guanidine hydrochloride or urea. Chaotropes should be avoided whenever possible since they are likely to denature the eluted protein. Methods for protein’s elution Methods 3 and 4: Specific elution by addition of a substance that competes for binding. These methods can enhance the specificity of media that use group-specific ligands.

Competitive elution Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand. And if our protein doesn’t have a known ligand? Fusion proteins and tags His tag

Kd coefficient AC purification steps

1. Equilibrate column for the specific purification process. 2. Load and bind protein. 3. Wash the column 5-10 CV 4. Elute. Equilibrate column for the specific purification process

20 mM TRIS pH 7.5 Similar to 200 mM NaCl Your lysis buffer 10-40mM Imidazole Loading column with protein solution Your lysis buffer+protein Protein sample is loaded and 20 mM TRIS pH 7.5 only protein containing the tag/binding site will be bound 200-300 mM NaCl to the column. The rest of 10-40mM imidazole (trial&error)* the proteins will elute as soon as the sample is injected Column wash

20 mM TRIS pH 7.5 Washing removes non- specific binders and low 200-1000 mM NaCl binding proteins. 20-100 mM Imidazole Elution

בשלב זה אנו מזרימים דרך mM TRIS pH 7.5 20 הקולונה את בופר האלוציה. אנו 50-200 mM NaCl משחררים את החלבון הקשור על ידי הוספת ריכוז גבוה של הליגנד. 300-500mM Imidazole בשלב זה יש לאסוף דוגמאות על מנת לזהות את החלבון מאוחר יותר.

GST-Glutathione S-transferase

26.4 kDa GST-Glutathione S-transferase

Protocol 1. Add 5 ml of a 50:50 slurry of glutathione-Sepharose beads to a lysate of 5gr of cells pellet (assuming expression is medium). Buffer: PBS or equivalent supplemented with 5 mM of BME or DTT (2mM TCEP). 2. Incubate 30’ at 4C rotating. 3. Collect flow through. 4. Elute in the presence of freshly prepared 10-20mM reduced glutathione. 5. If yield is low you might try increasing to 50 mM of reduced glutathione Maltose binding protein (MBP)

Cross linked Amylose beads

Elution with 10mM Maltose

40.6 kDa