ITC TroubleshootingWhat can go wrong?

Gamze KARAKULLUKÇU, MScApplication Engineer

gamze@atomikateknik.com | +90 549 745 30 04

ITC Troubleshooting

The Symptoms:

Baseline position

Baseline drift

Non sigmoidal binding isotherm

Stoichiometry far from 1 or integer value

Split (oscillating) peaks

Heat to Sample Cell = Negative Baseline Deflection

Heat to Reference Cell = Positive Baseline

Deflection

Cooling the Sample Cell = Positive Baseline

Deflection

Cooling the Reference Cell = Negative Baseline

Deflection

0

5

DP

DP

Temperature effects

Some instrumental factors

1. Lab conditions

2. Syringe

3. Stir speeds

4. Temperature setting

5. Plunger tip

0

5

DP

DP

Lab condition influence

0

5

DP

DP

Bent syringe

0

5

DP

MicroCal iTC200 - Syringe holding nut loose

DP

Syringe height

0

5

DP

Faster Stirring

Stir speeds

10 20 30 40 50 60 70

9.8

10.0

10.2

10.4

10.6

10.8

Ba

se

line

Po

sitio

n W

ith

Stirr

ing

(u

ca

l/se

c.)

Experimental Temperature (Deg. C)

Cell848

Cell849

Cell855

Cell857

Cell859

Cell860

Cell862

Cell864

Cell865

Cell866

Cell868

Cell869

###

###

Reference Power = 11 ucal/sec for all data shown below!

Baseline temperature dependence

Reference power=11 ucal/sec for all data shown

below!

Non instrumental factors

Baseline position is the first diagnostic for data

quality-information on:

Air bubbles

Cell cleanliness/“Sticky” proteins

Time between injections

0

5

DP

DP

Bubbles

0.00 20.00

9.6

9.8

10.0

Time (min)

µc

al/

se

c

De-gassing problem

0.00 20.008.28.38.48.58.68.78.88.99.09.19.29.39.49.59.69.79.89.9

Time (min)

µc

al/se

c

Before experiment:

De-gas, use proper filling technique (MicroCal iTC200)

During experiment:

Reload

Gently tap the bottom of the cell with the loading syringe

Re-insert injection syringe

The Cure

-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)

µca

l/se

c

Not long enough between injections

Time (min)

mca

l/s

0.00 33.33 66.67 100.00 133.33 166.67

14

15

16

17

18

19

Time (min)

µca

l/se

c

Not long enough between injections

Time (min)

mca

l/s

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)

The Cure

“Sticky” proteins or cleanliness

0.00 8.33 16.67 25.00 33.33 41.67 50.00

-4

-2

0

2

4

6

Time (min)

µca

l/se

c

-16.67 0.00 16.67 33.33 50.00 66.67 83.33 100.00 116.67 133.33

14

16

Time (min)

µcal/sec

-33.33 0.00 33.33 66.67 100.00 133.33 166.67 200.00 233.33

10

12

14

16

Time (min)

µcal/sec

0.00 16.67

9.38

9.40

9.42

9.44

9.46

9.48

MESdegasb98RAW_CP

MESdegasb98BASE

MESdegasb113RAW_CP

MESdegasb113BASE

Time (min)

µcal/sec

Time (min)

mcal/

s

Dirty cell

Clean the cells with Contrad 70 (Decon 90) and repeat

If the problem persists after cleaning then may have to

“do biochemistry”

Change solution conditions e.g. pH, salt, co-solutes

Change protein construct

The cure

0.00 10.00 20.00 30.009.10

9.20

9.30

9.40

Data1RAW_cp

###

Time (min)

µcal/sec

Nice data but dirty cell

Examine effect on stoichiometry and if error is not

acceptable:

Reload

Clean cell

The cure

Non sigmoidal binding isotherm

No Binding

No Heat

Buffer mismatch

More than one binding event

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 ml DMSO added to 1.0 ml buffer; cell: buffer only (no DMSO).

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

cell: 280 ml DMSO added to 14 ml buffer.

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

Time (min)

µcal/sec

2% DMSO into

2% DMSO-same stock

2% DMSO into

2% DMSO-separate stocks

2% DMSO into

0% DMSOm

ca

l/s

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)

µca

l/se

c

The cure

Dialyze, or use desalting columns if possible

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 mM and above-weak binding

-16.67 0.00 16.67 33.33 50.00 66.67 83.33 100.00 116.67 133.33 150.00

7.5

8.0

8.5

9.0

Experiment 2:

ligand into protein 2

Time (min)

µca

l/se

c

Control: ligand

into buffer

Experiment 1:

ligand into protein

1

No

saturation

or protein

not

matched

to ligand

Effect of buffer mismatch and/or low C

value

Time (min)

mca

l/s

Effect of buffer mismatch and/or low C

value

-16.67 0.00 16.67 33.33 50.00 66.67 83.33 100.00 116.67 133.33

29.0

29.5

30.0

30.5

31.0

31.5

Time (min)

µca

l/se

c

Time (min)

mca

l/s

See “mismatch” plus binding may be weaker than you

think

Increase sample concentrations

The cure

ITC: low heat

Heats for

experiment

same as control

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)

µcal/sec

Control: ligand into buffer

ExperimentL ligand into protein

Change experimental temperature by at least 10ºC

and/or

Increase sample concentration

The cure

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)

µca

l/se

c

Increase protein concentration or decrease ligand

concentration

Make sure protein is active

Also –c-value is high so we may want to change initial

[Lig]/[Prot] ratio

The cure

Split (oscillating) peaks

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33

-4

-2

0

2

Time (min)

µca

l/se

cOscillating

signal:

Power below 0

Time (min)

mcal/s

Instrument reference power too low

Clean sample and reference cell and increase ‘reference

power’ in advanced experimental tab

Note that ITC data can have exothermic and endothermic

peaks in same injection.

The cure

Insoluble Ligands

‘One site’ interactions are symmetrical and as such the

ligand can be put in the cell and the protein in the syringe

Reverse titration

-1.5

-1.0

-0.5

0.0

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

Time (min)

µca

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 in

jecta

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)

µca

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 in

jecta

nt

Parameter Ligand in syringe Ligand in cell

N 0.99 0.97

KD 104 nM 105 nM

DG -9.4 kcal/mol -9.4 kcal/mol

DH -11.9 kcal/mol -12.4 kcal/mol

TDS -2.5 kcal/mol -3.0 kcal/mol

29.5µM Protein titrated with

1.1mM Compound

11.5µM Compound titrated with

179µM Protein

Cure-reverse the titration

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

Stoichiometry not expected value

If stoichiometry less than expected value:

Protein concentration lower than you think

Ligand concentration higher than you think

Impure ligand and/or macromolecule

Misfolded protein (esp. for recombinants)

If 0.5, 1 ligand binding to 2 macromolecules (dimer?)

Binding does not fit simple independent model

Stoichiometry not expected value

If stoichiometry greater than expected value

Multiple binding sites on protein

Protein concentration higher than you think

Ligand concentration lower than you think

Binding does not fit simple independent model

ITC general conclusions!

Take care with sample preparation

Use appropriate run parameters

Check initial baseline position

Perform water-water titration to remove instrumental issues

De-gassing

Load carefully

Clean cell thoroughly, Clean cell thoroughly, Clean cell thoroughly