C.B.S. Scientific HTLE-7002 - Manual (Page 28)

C. B. S. œ Scientific 28 HTLE-7002
3.5 Phosphoamino Acid Analysis—continued
To load the samples follow the same procedure used for loading tryptic digest samples: I)
centrifuge the sample prior to loading to remove particulate debris, ii) load a spot of marker by at
the top of the plate, iii) load the samples by adding small drops using an Oxford micropipetors
and drying between each drop. You need only a few (between 10-100) cpm for each PAA. We
have detected phosphoamino acids in samples where no cpm could be detected using a counter!
The plate is now ready for electrophoresis. For larger sample volumes or loads one can also
spot the sample on a line using the protocol at the end of Section IIId.
Set up the electrophoresis apparatus as outlined in section IIId using pH1.9 buffer. The plates
are to be wetted using a blotter with 5 holes in it (corresponding to the 4 sample origins and 1 for
the dye marker) that has been dampened with 1.9 buffer. Wet the plate by overlaying the blotter
and squeeze out the buffer from the blotter around each of 5 holes as quickly as possible.
Remove the blotter and place the plate in the apparatus. Electrophoresis is carried out for 20 min.
at 1.5kV. Remove the plate when it is done and let it air dry using a fan for at least 20 min. Be
sure that the plate does not smell like acetic acid.
Change the buffer in the apparatus to pH3.5 by washing out the pH1.9 from each tank and
replacing with new wicks. Recently we have been adding EDTA to the pH3.5 buffer used for
wetting the blotters (0.5mM final). This appears to prevent streaking in the second dimension.
After the plate is completely dry from the first dimension you need to blot it with pH3.5 buffer for
the second dimension. To do this use strips of Whatman 3MM paper that are custom designed
for wetting each part of the plate (3cm wide for the bottom, 6.5cm wide for the middle, 10 cm wide
for the top)(see Figure in the Appendix). Dampen the three blotters in pH3.5 buffer plus EDTA.
Place these on the plate in rows parallel to the first dimension of separation about 1cm from the
plane of origin. Press along the edges to squeeze buffer out of wicks to wet the plate so the
buffer from two adjacent edges meets along the line of the origin to concentrate the sample for
the second dimension (see diagram in the Appendix). An additional piece of dampened blotting
paper, or gloved fingers dipped in buffer can be used to dampen any dry areas to make sure that
the plate is completely wet. Remove the blotters and soak up any puddled buffer with a
KimWipe. Place the plate in the apparatus making sure to rotate the plate 90
°C
counterclockwise. Electrophorese the plate for 16min. at 1.3kV. Disassemble the apparatus,
remove the plate and let it air dry using a fan for 20 min. as before or bake in the oven at 65
°C for
10min.
To visualize the cold phosphoamino acid spray the plate with 0.25% ninhydrin in acetone. Return
the plate to the 65
°C oven for at least 15 min. to develop the stain. Distinct purple spots will
appear that correspond to the positions of phosphoserine, phosphothreonine and
phosphotyrosine (see diagram in the Appendix). The PAA is complete and is now ready for
autoradiography.
Recently, Mark Kamps has shown that phosphoamino acids can be recovered from P
32
-labeled
proteins bound to PVDF membranes (Immobilon) after western transfer from SDS-
polyacrylamide gels(5). To do this simply immerse the piece of Immobilon membrane in constant
boiling HCl and hydrolyze as described above. When complete, remove the remaining liquid and
transfer to a new tube and lyophilize. The dried sample is treated using the same protocol as
described above. Note, nitrocellulose membranes cannot be treated with concentrated acid,
because they char and dissolve. Instead, one could first solubilize proteolytic peptides from the
membrane and perform PAA’s on the eluted material. Alternatively, the membrane bound protein
could be eluted directly with pyridine (2).