8. If the sample is aimed for MS analysis, extra care should be
taken to minimize keratin contamination from the surrounding
environment, and all samples, buffers, and tubes should be
handled only using gloves.
9. To avoid discrepancies between experiments, lysis buffer containing all the inhibitors can be aliquoted in 10 mL and stored
at À20
C. Aliquots are then thawed immediately before the
experiment.
10. This could represent a good stopping point as the cells can be
stored in liquid nitrogen or at À80
C for several weeks without any decrease in protein complex isolation.
11. We recommend keeping an aliquot of protein lysates from each
step to calculate the purification yield. This can be crucial for
some proteins known to be difficult to solubilize. Determining
the purification yield can help in the choice of detergent and
concentration to maximize the solubilization of your protein of
interest.
12. To avoid protein precipitation on the beads, we recommend
avoiding drying off the beads during the washing steps.
13. Peptides coupled to beads could be stored for at least 1 year in
PBS 20% ethanol at 4
C.
14. Streptavidin could be released during later steps from the beads
(boiling, etc.) and could interfere during MS analysis as a major
contaminant.
15. Longer peptides can be synthesized but price and purity can be
a limit.
16. The reaction is rapid and usually finished after 2 h incubation.
17. For longer term storage at 4
C, 20% ethanol in PBS was used.
18. The lysis step is critical and should be carefully addressed since
proteins should be extracted from cellular compartments
(nucleus, membrane, intracellular compartments) using detergents but the integrity of protein complexes should not be
altered by using too stringent conditions.
19. Volume of lysates used is dependent on the biological materials. 1 mL of 1 mg/mL protein lysate is a good starting point.
Abundance of interactors can be checked by silver staining. If
the abundance of the interactors is considered not to be sufficient, 1 mL at 5 mg/mL could be used or if needed using
larger volume in 15-mL conical tubes. Increase the volume of
beads will generate more background.
20. Preclearing with streptavidin beads is necessary to remove
biotin dependent carboxylases including acetyl-CoA carboxylase, propionyl CoA carboxylase considered as contaminants.
Identification of Associated PDZ Proteins
37
taken to minimize keratin contamination from the surrounding
environment, and all samples, buffers, and tubes should be
handled only using gloves.
9. To avoid discrepancies between experiments, lysis buffer containing all the inhibitors can be aliquoted in 10 mL and stored
at À20
C. Aliquots are then thawed immediately before the
experiment.
10. This could represent a good stopping point as the cells can be
stored in liquid nitrogen or at À80
C for several weeks without any decrease in protein complex isolation.
11. We recommend keeping an aliquot of protein lysates from each
step to calculate the purification yield. This can be crucial for
some proteins known to be difficult to solubilize. Determining
the purification yield can help in the choice of detergent and
concentration to maximize the solubilization of your protein of
interest.
12. To avoid protein precipitation on the beads, we recommend
avoiding drying off the beads during the washing steps.
13. Peptides coupled to beads could be stored for at least 1 year in
PBS 20% ethanol at 4
C.
14. Streptavidin could be released during later steps from the beads
(boiling, etc.) and could interfere during MS analysis as a major
contaminant.
15. Longer peptides can be synthesized but price and purity can be
a limit.
16. The reaction is rapid and usually finished after 2 h incubation.
17. For longer term storage at 4
C, 20% ethanol in PBS was used.
18. The lysis step is critical and should be carefully addressed since
proteins should be extracted from cellular compartments
(nucleus, membrane, intracellular compartments) using detergents but the integrity of protein complexes should not be
altered by using too stringent conditions.
19. Volume of lysates used is dependent on the biological materials. 1 mL of 1 mg/mL protein lysate is a good starting point.
Abundance of interactors can be checked by silver staining. If
the abundance of the interactors is considered not to be sufficient, 1 mL at 5 mg/mL could be used or if needed using
larger volume in 15-mL conical tubes. Increase the volume of
beads will generate more background.
20. Preclearing with streptavidin beads is necessary to remove
biotin dependent carboxylases including acetyl-CoA carboxylase, propionyl CoA carboxylase considered as contaminants.
Identification of Associated PDZ Proteins
37