ITC200 Training PDF

Isothermal titration calorimetry: Principles and experimental design

Stoyan Milev, Ph.D GE Healthcare [email protected]

2 GE Title or job number

1/9/2013

Agenda

Overview of Isothermal Titration Calorimetry

ITC experimental design

Data analysis

Troubleshooting

What is isothermal titration calorimetry (ITC)

A direct measurement of the heat generated or absorbed when molecules interact


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ITC applications

Study interactions between:

Protein-small molecule

Enzyme-inhibitor

Protein-protein

Protein-DNA

Protein-lipid

Protein-carbohydrate

Etc.


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Microcalorimetry offers enhanced information content

Experimental biological relevance

Label-free

In-solution

No molecular weight limitations

Optical clarity unimportant

Minimal assay development


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How Do ITCs Work?

Reference Calibration Heater

Cell Main Heater

Sample Calibration Heater

DP

DT

S R The DP is a measured power differential between the reference and sample cells necessary to maintain their temperate difference at close to zero

DT~0


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Performing an ITC experiment Ligand in syringe

Macromolecule in sample cell

Heat of interaction is measured

Parameters measured from a single ITC experiment:

Affinity - KD

Energy (Enthalpy) - DH

Number of binding sites - n

Reference Cell Sample Cell

Syringe


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Ligand in syringe Macromolecule in ITC cell

ITC Before titration


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Ligand in syringe

Macromolecule in cell

Macromolecule-ligand complex

As the first injection is made, all injected ligand is bound to target macromolecule.

Titration begins: First injection

5

0


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The signal returns to baseline before the next injection.

Return to baseline

5

0


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As a second injection is made, again all injected ligand becomes bound to the target.

Second injection

5

0


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Second return to baseline

Signal again returns to baseline before next injection.

5

0


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As the injections continue, the target becomes saturated with ligand, so less binding occurs and the heat change starts to decrease.

Injections continue

5

0


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As the injections continue, the target becomes saturated with ligand so less binding occurs and the heat change starts to decrease.

Injections continue

5

0


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When the macromolecule is saturated with ligand, no more binding occurs, and only heat of dilution is observed.

End of titration

5

0


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Experimental results

= 1/KD

5

0


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MicroCal VP-ITC MicroCal iTC200 MicroCal Auto-iTC200

1400 L cell

Manual sample loading

Up to 5 samples/day

Sensitive

Fast

Easy to use

KD from mM to nM

200 L cell

Upgradable to full automation

Unattended operation

Up to 75 samples/day (using single injection method)

KD from mM to nM

Sample cell is 200 L

Easy to use

96-well plate format

MicroCal ITC systems

Why ITC?


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Stoichiometry

Number of ligand binding sites per macromolecule

If one binding site the stoichiometry is 1

By convention a Ligand has one binding site

A Macromolecule can have more than one binding site


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Effective Binding Affinity Range

KD in mM to nM range

Weak binding low C-value method

Tight binding - minimize injection volume and concentration or use competitive (displacement) binding procedure and fitting model


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Thermodynamics

KB binding constant

KD = 1/ KB =

DG = RT lnKD

DG = DH - T DS

`

[L] x [M]

[ML]


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Free energy change

DG is change in free energy

DG 0 for spontaneous process

More negative DG, higher affinity


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Enthalpy change

DH measure of the energy content of the bonds broken and created. The dominant contribution is from hydrogen bonds.

Negative value indicates enthalpy change favoring the binding

Solvents play a role


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Entropy change

DS positive for entropically driven reactions

Hydrophobic interactions

Solvation entropy (favorable) due to release of water

Conformational degrees of freedom (unfavorable)


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Microcalorimetry provides a total picture of binding energetics

H

-TS

Overall binding affinity KD correlates with IC50 or EC50. This is directly related to G, the total free binding energy

DG = DH -TDS

H, enthalpy is indication of changes in hydrogen and van der Waals bonding

-TS, entropy is indication of changes in hydrophobic interaction and conformational changes

N, stoichiometry indicates the ratio of ligand-to-macromolecule binding


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Same affinity, different energetics! All three interactions have the same binding energy (G) A. Good hydrogen bonding with

unfavorable conformational

change

B. Binding dominated by

hydrophobic interaction

C. Favorable hydrogen bonds

and hydrophobic interaction

ITC results are used to get insights into mechanism of binding

-20

-15

-10

-5

0

5

10

kc

al/

mo

le G

H

-TS

Favorable

Unfavorable

A B C

How to get good ITC data


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C Values

0.0 0.5 1.0 1.5 2.0

-16

-14

-12

-10

-8

-6

-4

-2

0

Molar Ratio

kcal/m

ole

of in

jecta

nt

0.05

0.5

5

50

500

C = [M]

KD

Example: Kd = 100nM [M] = 100nM, C=1 [M] = 5uM, C=50 [M]:[L] 1:10 for n=1


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-2

-1

0

0 30 60 90 120 150

(a)

tim e (m in)

c

al/

s

0.0 0.5 1.0 1.5 2.0 2.5 3.0

-16

-12

-8

-4

0

(b)

molar ratio (Ras / RalGDS-RBD)

kc

al/

mo

l o

f in

jec

ted

Ra

s

c = 5

c = 40

Kd = 1.2 M

C Values


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C Values in ITC

C = {[M]tot / KD} * N

C = 10-100 very good

C = 5-500 good

C = 1-5 and 500-1000 OK

C = < 1 and > 1000 not wanted


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ITC experimental design kc

al

mo

l-1 o

f in

jec

tan

t

Molar ratio

-4

-2

0

0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0

-4

-2

0

Molar Ratio

kca

l/m

ole

of in

ject

ant

10< [Protein]/KD > 1000

Fitted: N, DH

0.0 0.5 1.0 1.5 2.0

-4

-2

0

Molar Ratio

kca

l/m

ole

of in

ject

ant

-0.4

-0.2

0

0 4 8 12 16

[Protein]/KD < 1 N fixed

Fitted: KD, DH

High C Low C


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ITC experimental design

KD (Biacore) M [Protein] M [Compound] M [Protein] / KD

20

0.5-2 30 300 15-60

2-10 50 500 5-25

10-100 30 40*KD (Biacore) 0.3-3

>100 30 20*KD (Biacore)


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C Values-Low

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

-14

-12

-10

-8

-6

-4

-2

0

Molar Ratio

kcal/m

ole

of in

jecta

nt

0 2

-1.7

-1.6

-1.5

-1.4

-1.3

-1.2

-1.1

Molar Ratio

kcal/m

ole

of in

jecta

nt

Note different scales


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C Values-Low

Increase ligand concentration but not the valuable protein

0 2 4 6 8 10 12 14 16 18 20 22 24 26

-2

0

Molar Ratio

kcal/m

ole

of in

jecta

nt

0--------------->26


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Displacement ITC HIV-1 Protease- Inhibitor Binding

Amprenavir Acetyl pepstatin Amprenavir + acetyl pepstatin

Ohtaka, et al, Protein Sci. 11, 1908-1916 (2002)

Unable to determine KB KB of 3.1 x 1010 M-1

ITC practical considerations


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ITC practical considerations

0.00 10.00 20.00 30.00 40.00 50.00

3.15

3.20

3.25

3.30

3.35

3.40

3.45

3.50

3.55

protein A in syringe titrated into protein B

Time (min)

cal/sec

protein A in syringe titrated into buffer

0 1 2 3

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

2

Molar Ratio

kcal/m

ole

of in

jecta

nt


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MicroCal iTC200: Minimum Heat=Minimum [Protein]

For iTC200:

Average heat required per injection ~ 1 cal

Need ~ 10 injections

Average DH of binding is ~ -5 kcal/mol

Therefore minimum sample concentration, [Prot], required in a 200 l cell is

Total heat, Q, = 1 cal/injection * 10 injections = 10 cal Q = (cell volume) x (DH) x [Prot]

[Prot] = (10 cal)/(200 l)/(5,000 cal/moles)]

Min [Prot] ~ 10 M


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Ligand concentration

[L] = 10 to 20 x [M]

May need to be adjusted based on experiment

At end of ITC, for N of 1, final [L]/[M] ratio should be 2 to 4 to ensure saturation of all M binding sites


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iTC200 Experimental Design Tab


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With little prior knowledge

Good Starting Conditions

300M Ligand in the Syringe and

30M Macromolecule in the cell

16 x 2.5 l injections

Ideal for KDs of 1 M 100 nM


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Use dialysis or buffer exchange column

Check calibration pipettes-by weight

Retain the dialysate/exchanged buffer

Adopt and stick to a reproducible protocol

Sample preparation


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Choice of buffer

Buffers have ionization enthalpies:

BH B- + H+

Use buffers with DHion ~ 0

Including; phosphate, acetate, formate, citrate, sulfate, cacodylate, glycine

Quaternary amines (e.g. Tris) have high DHion

DHion


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Avoid DTT

Unstable and undergoes oxidation

High background heat

Use b-mercaptoethanol & TCEP

TCEP is not stable in phosphate buffer

Use conditions in which your protein is happy

Choice of buffer


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Buffer Mismatch-No Dialysis

0 20 40 60 80 100 120 140 160 180

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

without dialysis

with dialysis

Time (min)

cal/sec


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Sample preparation: small molecule ligand

If ligand is too small to dialyze, be sure material is desalted prior to final preparation

Use final dialysis buffer of macromolecule to dissolve ligand

Match pH


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iTC200 Experimental design

Injection volume recommended 1 to 3 l ((range 0.1-38 l)

Injection rate should be 0.5 l/sec

Spacing- typical 120 to 180 sec

Filter period - 5 sec or less recommended

Reference power typical 6 ucal/sec

High feedback for most ITC experiments

Stirring - 1000 rpm


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iTC200

Macromolecule (in the cell):

Need 300 l

Ligand (in the syringe):

Need 70 l


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ITC Enthalpy Changes

DHobserved by ITC is total of :

DHbinding

DHionization

DHconformation

Any non-specific effects (buffer mismatch, pH mismatch, heat of dilution, heat of ligand dissociation)

Need to account for these effects by appropriate controls and experimental conditions


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Controls Injection of syringe material into buffer-

Peaks should be similar in magnitude to those at the end of the actual titration experiment and constant

0 10 20 30 40 50 60 70 80 90

4.25

4.30

4.35

4.40

4.45

4.50

4.55

4.60

4.65

4.70

4.75

A into B

A into Buffer

Time (min)

cal/sec


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Buffer Mismatch

-5 0 5 10 15 20 25 30 35 40 45 50 55

0

1

2

3

4

5

6

7

C: 2 % mismatch in DMSO: syringe: 20 l DMSO added to 1.0 ml buffer; cell: buffer only (no DMSO).

B: Slightly mismatched solution: syringe: 20 l DMSO added to 1.0 ml buffer;

cell: 280 l DMSO added to 14 ml buffer.

A: Matched solution: both cell & syringe have same solution (280 l DMSO added to 14 ml buffer).

Time (min)

cal/sec

2% DMSO into 2% DMSO

2% DMSO into 1.95% DMSO



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Dissociation of Syringe Material into Buffer

Calorimetric dilution data showing the effects of different ligands on dilution of insulin

Ref: Lovatt M, Cooper A and Camillerri P (1996)

Eur. Biophys. J. 24:354-357

Troubleshooting


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Why Do ITC Experiments Not Work

as Expected? Concentration errors because KD is unknown or

different value than expected

Too little heat change

Never reach saturation

Rapidly saturate binding sites

Incorrect concentrations used

Buffer mismatch too large heat of dilution

Other sources of heat change in system

No binding


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The Symptoms

Baseline Position

Baseline Drift

Split (oscillating) peaks

Non sigmoidal binding isotherm

Stoichiometry far from 1 or integer value


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Bent Syringe

0

5

DP

DP


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Syringe Height

0

5

DP

iTC200 - Syringe Holding Nut Loose

DP


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Non Instrumental Factors


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Baseline Position/Drift

Baseline position is the first diagnostic for data quality-information on

Cell cleanliness

Sticky proteins

Air Bubbles

Time between injections


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Bubbles

0

5

DP

DP


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The Cure

Reload

Or

Gently tap the bottom of the cell with the loading syringe


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Not long enough between injections

0.00 33.33 66.67 100.00 133.33 166.67

14

15

16

17

18

19

Time (min)

ca

l/se

c


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Not long enough between injections

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 66.67

2

4

6

8

10

12

14

16

Time (min)

ca

l/se

c


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The Cure

Increase the time between the injections

This can be done on the fly by going to the advanced experimental tab and update the change to injection parameters>spacing(secs)


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Sticky Proteins or Cleanliness

-33.33 0.00 33.33 66.67 100.00 133.33 166.67 200.00 233.33 266.67

0

2

4

Time (min)

ca

l/se

c Change in baseline


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0.00 8.33 16.67 25.00 33.33 41.67 50.00

-4

-2

0

2

4

6

Time (min)

cal/sec


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-16.67 0.00 16.67 33.33 50.00 66.67 83.33 100.00 116.67 133.33

14

16

Time (min)

ca

l/se

c


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-33.33 0.00 33.33 66.67 100.00 133.33 166.67 200.00 233.33

10

12

14

16

Time (min)

ca

l/se

c


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Non sigmoidal binding isotherm

No Binding

No Heat

Buffer mismatch

More than one binding event


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Effect of DMSO Mismatch

-5 0 5 10 15 20 25 30 35 40 45 50 55

0

1

2

3

4

5

6

7

C: 2 % mismatch in DMSO: syringe: 20 l DMSO added to 1.0 ml buffer; cell: buffer only (no DMSO).

B: Slightly mismatched solution: syringe: 20 l DMSO added to 1.0 ml buffer;

cell: 280 l DMSO added to 14 ml buffer.

A: Matched solution: both cell & syringe have same solution (280 l DMSO added to 14 ml buffer).

Time (min)

cal/sec

2% DMSO into 2% DMSO-same stock

2% DMSO into 2% DMSO-separate stocks



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Buffer Mismatch-No Dialysis

0 20 40 60 80 100 120 140 160 180

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

without dialysis

with dialysis

Time (min)

cal/sec


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The Cure

Dialyze or use desalting column

Check for additives that are in not in both cell and syringe- Also-ask what was sample purified from e.g. was protein lyophilized in buffer and not dialyzed

Check pH of final solutions-should differ by less than 0.1 pH units. This issue is common when working with high concentrations of ligand-e.g. 500 M and above-weak binding


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ITC: low heat

0.00 16.67 33.33 50.00 66.67 83.33

4.26

4.28

4.30

4.32

4.34

4.36

4.38

4.40

4.42

4.44

4.46

4.48

4.50

4.52

Time (min)

ca

l/se

c

Control: ligand into buffer

ExperimentL ligand into protein

Heats for experiment same as control


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The Cure

Change experimental temperature by at least 10 C

AND/OR

Increase sample concentration


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Unexpected Stoichiometry

0.00 33.33 66.67 100.00 133.33 166.67

18.6

18.7

18.8

18.9

19.0

19.1

19.2

19.3

Time (min)

ca

l/se

c


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Split (oscillating) peaks


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Instrument reference power too low

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33

-4

-2

0

2

Time (min)

ca

l/se

c

Oscillating signal: Power below 0


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The Cure

Clean and increase reference power in advanced experimental tab


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Split Peaks-Sometimes its OK

ITC shows differential binding of Mn(II) ions to WT T5 5 nuclease

-2

0

2

4 -10 0 10 20 30 40 50 60 70 80 90 100

Time (min)

cal/sec

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

2

Molar Ratio

kca

l/m

ole

n = 0.85

Ka = 3.0 x 105 M-1

DH = -0.59 kcal mol-1

n = 1.3

Ka = 1.0 x 104 M-1

DH = +1.6 kcal mol-1

Feng, et al, Nat. Struct. Mol. Biol. 11, 450-456 (2004)


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Insoluble Ligands

One site interactions are symmetrical and as such the ligand can be put in the cell and the protein in the syringe


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-1.5

-1.0

-0.5

0.0

-10 0 10 20 30 40 50 60 70 80 90 100 110 120

Time (min)

ca

l/se

c

0.0 0.5 1.0 1.5 2.0 2.5

-12

-10

-8

-6

-4

-2

0

2

Molar Ratio

kca

l/m

ole

of

inje

cta

nt

-0.8

-0.6

-0.4

-0.2

0.0

-10 0 10 20 30 40 50 60 70 80 90 100

Time (min)

ca

l/se

c

0.0 0.5 1.0 1.5 2.0 2.5

-14

-12

-10

-8

-6

-4

-2

0

2

Molar Ratio

kca

l/m

ole

of

inje

cta

nt

Parameter

Ligand in syringe

Ligand in cell

n

0.99

0.97

Kd

104 nM

105 nM

DGo

-9.4 kcal/mol

-9.4 kcal/mol

DHo

-11.9 kcal/mol

-12.4 kcal/mol

TDSo

-2.5 kcal/mol

-3.0 kcal/mol

29.5M Protein titrated with 1.1mM Compound

11.5M Compound titrated with 179M Protein

Cure-Reverse the Titration


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Stoichiometry

N is the average number of binding sites per mole of protein in your solution, assuming:

that all binding sites are identical and independent

that you have pure protein (and ligand)

that you have given the correct protein and ligand concentrations

that all your protein is correctly folded and active


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ITC General Troubleshooting

Clean cell and syringe thoroughly

Take care with sample preparation

Check initial baseline position

Use appropriate run parameters

Perform water-water titration to rule out instrumental issues


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ITC Maintenance

Cell cleaning:

Water/buffer

20 % Contrad 70 soak for sticky proteins

Keep water in cell when not in use

Reference cell:

Replace water once a week


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ITC Maintenance

Syringe:

20 % Contrad 70 soak for sticky proteins

Store dry

Do not overtighten fill port adaptor

Take care to not bend needle or crack syringe

Change plunger tip when needed


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ITC200 Training PDF