7. Using gradient gels with increasing acrylamide concentration
from top to bottom usually 4–12% allows the separation and
visualization of large proteins (200 kDa) that can still enter the
gel at the top and small (10 kDa) polypeptides such as PDZ
domains on the same gel as they retain small and fast-moving
proteins in the bottom of the gel.
8. Proteins larger than 800 kDa will not enter in the
polyacrylamide gel.
9. Check that there is no air bubble in the cuvette.
10. Record a spectrum over a wide wavelength range with a wavelength as a starting point where the solute does not absorb.
11. HCCA, DHB, and sDHB for PDZ samples with molecular
masses below 20 kDa and sinapinic acid is usually used for
constructs encompassing PDZ domain above 20 kDa. However, sinapinic acid produces adducts that may impede the
resolution and the precision of the m/z. In this case, the
HCCA matrix could be a good alternative for large
PDZ-containing constructs.
If the buffer is supplemented with more than 1% glycerol
or detergent, the best practice is to concentrate the sample and
then dilute it in water to reach the concentration required, to
minimize the effect of the detergent or glycerol. Sometimes,
the concentration of the purified PDZ is below what is required
to perform the MALDI-TOF analysis. Concentration and
desalting of PDZ constructs of molecular masses lower than
15 kDa could be achieved on a ZipTip C18. ZipTip C4 is used
for PDZ constructs of higher molecular mass.
12. High laser power triggers the loss of resolution and precision
on the m/z.
13. A database of the most common protein post translation modifications is found here: https://abrf.org/delta-mass
14. Between 25 and 50 pmol of PDZ are required. In case of
quantity limitation, use a ZipTip to desalt and concentrate
the protein. Keep in mind that the PDZ solution must be
pure to avoid false positive.
15. To remove dust or salt crystals, the buffer must be filtered.
16. For quality control, we do not recommend to filter or centrifuge the sample in order to detect aggregates; if DLS is used for
other purposes, it is advisable to filter the sample and centrifuge it for 5 min in a benchtop centrifuge at full speed before
loading.
17. Care should be taken to choose the column as an elongated
10 kDa protein may behave on the column like a globular
35 kDa protein. Be aware that SEC columns are often
described by their separative power on globular protein.
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from top to bottom usually 4–12% allows the separation and
visualization of large proteins (200 kDa) that can still enter the
gel at the top and small (10 kDa) polypeptides such as PDZ
domains on the same gel as they retain small and fast-moving
proteins in the bottom of the gel.
8. Proteins larger than 800 kDa will not enter in the
polyacrylamide gel.
9. Check that there is no air bubble in the cuvette.
10. Record a spectrum over a wide wavelength range with a wavelength as a starting point where the solute does not absorb.
11. HCCA, DHB, and sDHB for PDZ samples with molecular
masses below 20 kDa and sinapinic acid is usually used for
constructs encompassing PDZ domain above 20 kDa. However, sinapinic acid produces adducts that may impede the
resolution and the precision of the m/z. In this case, the
HCCA matrix could be a good alternative for large
PDZ-containing constructs.
If the buffer is supplemented with more than 1% glycerol
or detergent, the best practice is to concentrate the sample and
then dilute it in water to reach the concentration required, to
minimize the effect of the detergent or glycerol. Sometimes,
the concentration of the purified PDZ is below what is required
to perform the MALDI-TOF analysis. Concentration and
desalting of PDZ constructs of molecular masses lower than
15 kDa could be achieved on a ZipTip C18. ZipTip C4 is used
for PDZ constructs of higher molecular mass.
12. High laser power triggers the loss of resolution and precision
on the m/z.
13. A database of the most common protein post translation modifications is found here: https://abrf.org/delta-mass
14. Between 25 and 50 pmol of PDZ are required. In case of
quantity limitation, use a ZipTip to desalt and concentrate
the protein. Keep in mind that the PDZ solution must be
pure to avoid false positive.
15. To remove dust or salt crystals, the buffer must be filtered.
16. For quality control, we do not recommend to filter or centrifuge the sample in order to detect aggregates; if DLS is used for
other purposes, it is advisable to filter the sample and centrifuge it for 5 min in a benchtop centrifuge at full speed before
loading.
17. Care should be taken to choose the column as an elongated
10 kDa protein may behave on the column like a globular
35 kDa protein. Be aware that SEC columns are often
described by their separative power on globular protein.
120
Ce ´ lia Caillet-Saguy et al.