dry overnight. If leaving sample overnight, do not spot calibration standard on the plate until ready to analyze in order to
prevent degradation of the standard.
17. This procedure is best suited for a purified protein concentrate
(100 μM) soluble in an aqueous buffer with no detergents
(such as PBS).
18. This procedure is best suited for a purified protein in solution
where the protein is either very dilute, in a solution containing
detergents, or poorly soluble.
19. Other precipitation methods such as protein precipitation with
acetone has been proven to be suitable for protein samples with
detergents or lipid contaminants; this may result in the formation of additional protein adducts [29]. The combination of
trichloroacetic acid (TCA) to acetone is also recommended
where acetone insoluble but TCA soluble contaminants are
present in the sample [30]. Other precipitation methods can
be found in Refs. [31, 32].
20. If the protein sample is dry, reconstitute in 10–100 mM ammonium acetate prior to addition of organic solvent.
21. Alternatively, dilutions of the precipitated protein can be
prepared as in Subheading 3.2.
22. This procedure is best suited for a purified protein in solution
where the protein is either very dilute or in a solution with a
high salt concentration or other ion suppressing minerals.
23. Volume of ammonium acetate will vary depending on the size
of the ultrafiltration device. It is recommended to use at least
half of the volume size. For instance, for a 0.5 mL device, add
0.25 mL, for a 4 mL device, add 2 mL, and for a 15 mL device,
add 7.5 mL.
24. Also suitable for analysis via Multiple Reaction Monitoring
(MRM).
25. Gel loading pipet tips or other small caliber tips are recommended in order to prevent gel pieces from being picked up
during pipetting.
26. Gel pieces will begin to dehydrate during this step, which can
sometimes interfere with destaining. After the second round of
destaining, if the gel still contains a significant amount of
visible stain, or if the gel pieces start to turn white (indicating
dehydration), rehydrate as follows:
(a) Add 100 μL of 0.2 M ammonium bicarbonate;
(b) Allow gel pieces to reswell for 15 min;
(c) Remove excess ammonium bicarbonate;
(d) Add destain and proceed with incubation;
Proteomic Analyses
545
prevent degradation of the standard.
17. This procedure is best suited for a purified protein concentrate
(100 μM) soluble in an aqueous buffer with no detergents
(such as PBS).
18. This procedure is best suited for a purified protein in solution
where the protein is either very dilute, in a solution containing
detergents, or poorly soluble.
19. Other precipitation methods such as protein precipitation with
acetone has been proven to be suitable for protein samples with
detergents or lipid contaminants; this may result in the formation of additional protein adducts [29]. The combination of
trichloroacetic acid (TCA) to acetone is also recommended
where acetone insoluble but TCA soluble contaminants are
present in the sample [30]. Other precipitation methods can
be found in Refs. [31, 32].
20. If the protein sample is dry, reconstitute in 10–100 mM ammonium acetate prior to addition of organic solvent.
21. Alternatively, dilutions of the precipitated protein can be
prepared as in Subheading 3.2.
22. This procedure is best suited for a purified protein in solution
where the protein is either very dilute or in a solution with a
high salt concentration or other ion suppressing minerals.
23. Volume of ammonium acetate will vary depending on the size
of the ultrafiltration device. It is recommended to use at least
half of the volume size. For instance, for a 0.5 mL device, add
0.25 mL, for a 4 mL device, add 2 mL, and for a 15 mL device,
add 7.5 mL.
24. Also suitable for analysis via Multiple Reaction Monitoring
(MRM).
25. Gel loading pipet tips or other small caliber tips are recommended in order to prevent gel pieces from being picked up
during pipetting.
26. Gel pieces will begin to dehydrate during this step, which can
sometimes interfere with destaining. After the second round of
destaining, if the gel still contains a significant amount of
visible stain, or if the gel pieces start to turn white (indicating
dehydration), rehydrate as follows:
(a) Add 100 μL of 0.2 M ammonium bicarbonate;
(b) Allow gel pieces to reswell for 15 min;
(c) Remove excess ammonium bicarbonate;
(d) Add destain and proceed with incubation;
Proteomic Analyses
545