Step
Problem
3
Unstable ITC baseline Sample cell and syringe are
or baseline shift
dirty
Air bubbles in the sample
cell
13
Absence of a PCR
product after fusion
PCR
Multiple
PCR
products after fusion
PCR
No protein in elution
17
18
No
protein
peak
observed in the elution
profile
18
Protein eluted in void
volume
Possible Reasons
Solution
Thoroughly wash the cell and
syringe
Remove air bubbles. Take care not
to introduce bubbles when filling
the cell and syringe
Fusion of gene did not take Recheck the primers and repeat
place
the reaction by slightly varying
the annealing temperature
The primers may have bound Redesign the primers and repeat
to
related
sequences the reaction
elsewhere in the template
Cell culture conditions are Try
different
cell
culture
not suitable
conditions by varying the IPTG
concentration and temperature for
induction and induction period
Protein is in the insoluble Use a refolding method or use a
fraction
solubility tag to make the protein
soluble
No binding with Ni-NTA Use fresh Ni-NTA resin and
resin
equilibrate with lysis buffer for a
minimum of 1 h
Protein eluted away during Remove imidazole from wash
wash steps
buffers and repeat the assay
Protein has leaked due to Check the connections of the loop.
tubing connections.
Try to reconnect and inject the
protein
UV lamp is not sensitive; Change the UV lamp for better
this is particularly a concern sensitivity
if the protein concentration is
low
Protein shows higher order Optimize buffer conditions to
oligomerization
prevent aggregation
Binding partners may not be Optimize the length of the linker
interacting to form an intact to retain natural interactions
complex
between the binding partners