beer-lambert law - bgulifeserv.bgu.ac.il/wb/zarivach/media/ppc/tutorials/exercise 5-ac_en... ·...
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![Page 1: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/1.jpg)
Beer-Lambert Law
Beer-Lambert Law:
log(I0/I) = ebc
e = A/cb
A = ebc
A = ec (when b is 1 cm)
I0 = intensity of incident light
I = intensity of transmitted light
e = molar absoptivity coefficient in cm2
mol-1
c = concentration in mol L-1
b = pathlength of absorbing
solution in cm-1
A = absorbance = log(Io/I)
0.1
cm
ℓ
http://www.hellma-worldwide.de/en/default.asp
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For example…
• Protein abs @ 280 (1g/L) = 1.0
• Abs is 0.67
• Thus – Abs/abs(1g/L)=0.67mg/ml
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Protein purification and characterization
Exercise #5
Affinity Chromatography
![Page 4: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/4.jpg)
Protein Purification & Characterization
![Page 5: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/5.jpg)
What is Chromatography?Invented by a Russian botanist named Mikhail Tswett in 1905. He separated plant pigments using glass columns packed with calcium carbonate.
![Page 6: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/6.jpg)
Before we proceed…
FPLC (AKTA Purifier) Gravity-assisted glass column
How we conduct chromatography?
![Page 7: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/7.jpg)
עקרונות ההפרדהAffinity 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?
![Page 8: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/8.jpg)
Customization of affinity column
• Choosing the right ligand (verify dissociation)
• Choose the right matrix:
– Hydrophilic
– Large spaces
– Rigid
– Inert
– Chemically stable
• Attaching ligand to matrix
For example, immobilization of IgG to agarose beads
Aldehyde
activation
![Page 9: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/9.jpg)
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.
![Page 10: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/10.jpg)
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
![Page 11: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/11.jpg)
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Method 1:The simplest case. A change of buffer composition elutes the bound substance without harming either it or the ligand.
pH elutionA 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 elutionThe 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
![Page 13: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/13.jpg)
Method 2:Extremes of pH or high concentrations of chaotropic agents are required for elution, but these may cause permanent or temporary damage.
Chaotropic eluentsIf other elution methods fail, deforming buffers, which alter the structure of proteins, canbe 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
![Page 14: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/14.jpg)
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 elutionSelective 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.
Methods for protein’s elution
![Page 15: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/15.jpg)
And if our protein doesn’t have a known ligand?
Fusion proteins and tags
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His tag
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Kd coefficient
![Page 19: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/19.jpg)
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.
![Page 20: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/20.jpg)
Equilibrate column for the specific purification process
20 mM TRIS pH 7.5
200 mM NaCl
10-40mM Imidazole
Similar to
Your lysis buffer
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Loading column with protein solution
Protein sample is loaded and
only protein containing the
tag/binding site will be bound
to the column. The rest of
the proteins will elute as soon
as the sample is injected
20 mM TRIS pH 7.5
200-300 mM NaCl
10-40mM imidazole (trial&error)*
Your lysis buffer+protein
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Column wash
Washing removes non-
specific binders and low
binding proteins.
20 mM TRIS pH 7.5
200-1000 mM NaCl
20-100 mM Imidazole
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Elution
בשלב זה אנו מזרימים דרך
אנו . הקולונה את בופר האלוציה
משחררים את החלבון הקשור על
. ידי הוספת ריכוז גבוה של הליגנד
בשלב זה יש לאסוף דוגמאות על
מנת לזהות את החלבון מאוחר
.יותר
20 mM TRIS pH 7.5
50-200 mM NaCl
300-500mM Imidazole
![Page 24: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/24.jpg)
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GST-Glutathione S-transferase
26.4 kDa
![Page 26: Beer-Lambert Law - BGUlifeserv.bgu.ac.il/wb/zarivach/media/PPC/Tutorials/Exercise 5-AC_EN... · Beer-Lambert Law Beer-Lambert Law: log(I 0 /I) = ebc e= A/c b A = ebc A = ec ... Affinity](https://reader031.vdocument.in/reader031/viewer/2022021506/5b0267437f8b9a54578fa5cb/html5/thumbnails/26.jpg)
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 4C 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
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Maltose binding protein (MBP)
40.6 kDa
Cross linked Amylose beads
Elution with 10mM Maltose