8. Remove the medium gently and add 100 μL of DMSO to the
wells.
9. Shake the plate at 400 rpm for 15 min at 50
C to dissolve the
formazon product.
10. Analysis by ELISA microplate reader at 570 nm. The cell
viability is expressed in a percentage of A 570 nm values of the
average of the sample vials to the control wells. These are
exposed to HBSS in the absence of any drug treatment as the
treatment of 5 mM sodium azide leads to complete cell death
(see step 3)
4 Notes
1. If the DP value is not close to the reference DP value in Æ0.5
difference, there is a strong possibility that the peptide/protein
is aggregating inside the cell, there is an air bubble trapped or
that there are some dust particles inside.
2. Wang resin is being used as it comes with an acid linker and it
can be bought preloaded with the first amino acid. As an
alternative, chlorotrityl resin can be easily used. This resin
provides an acid linker and also removes any possible side
chain racemization due to the steric bulk trityl groups.
3. The scale of the synthesis means the amount of resin that is
required to obtain a specific amount of crude peptide.
The amount of resin needed to achieve a determined scale is
determined by the loading of the resin usually provided as
mmol g
À1 .
4. Here we just describe standard coupling reagents such as
HBTU; however, the reader should be aware that depending
on the difficulty of the synthesis, other more reactive coupling
reagents such as the phosphonium base salt PyAOP can
be used.
5. The Kaiser test usually just gives as an indication the blue color;
however, depending on the sequence and the amino acid, it is
possible to observe different bead colorations not stated in the
test. In such cases, we suggest to do a double coupling and if
there is no change on the color, continue working with
it. Alternatively, perform a test cleavage.
6. It is recommended to plan test cleavages along the synthesis
(specially for long sequences) as it will provide crucial information on which amino acids are more difficult or problematic to
couple. We strongly suggest to perform a test cleavage when
the peptide is completed as a mandatory step as it will provide
information on the peptide quality and in worst-case scenario,
if it is required to restart the synthesis.
174
Dominik J. Essig et al.
wells.
9. Shake the plate at 400 rpm for 15 min at 50
C to dissolve the
formazon product.
10. Analysis by ELISA microplate reader at 570 nm. The cell
viability is expressed in a percentage of A 570 nm values of the
average of the sample vials to the control wells. These are
exposed to HBSS in the absence of any drug treatment as the
treatment of 5 mM sodium azide leads to complete cell death
(see step 3)
4 Notes
1. If the DP value is not close to the reference DP value in Æ0.5
difference, there is a strong possibility that the peptide/protein
is aggregating inside the cell, there is an air bubble trapped or
that there are some dust particles inside.
2. Wang resin is being used as it comes with an acid linker and it
can be bought preloaded with the first amino acid. As an
alternative, chlorotrityl resin can be easily used. This resin
provides an acid linker and also removes any possible side
chain racemization due to the steric bulk trityl groups.
3. The scale of the synthesis means the amount of resin that is
required to obtain a specific amount of crude peptide.
The amount of resin needed to achieve a determined scale is
determined by the loading of the resin usually provided as
mmol g
À1 .
4. Here we just describe standard coupling reagents such as
HBTU; however, the reader should be aware that depending
on the difficulty of the synthesis, other more reactive coupling
reagents such as the phosphonium base salt PyAOP can
be used.
5. The Kaiser test usually just gives as an indication the blue color;
however, depending on the sequence and the amino acid, it is
possible to observe different bead colorations not stated in the
test. In such cases, we suggest to do a double coupling and if
there is no change on the color, continue working with
it. Alternatively, perform a test cleavage.
6. It is recommended to plan test cleavages along the synthesis
(specially for long sequences) as it will provide crucial information on which amino acids are more difficult or problematic to
couple. We strongly suggest to perform a test cleavage when
the peptide is completed as a mandatory step as it will provide
information on the peptide quality and in worst-case scenario,
if it is required to restart the synthesis.
174
Dominik J. Essig et al.