Biomedical Engineering Reference
In-Depth Information
30. For an 8 cm by 10 cm membrane, a minimum volume of 3 mL
of antibody diluent buffer solution is needed. The 5 % nonfat
milk in PBST buffer is used for both blocking and diluting the
primary and secondary antibodies for immunoblotting.
31. Depending on the strength of the signal, you may need to
dilute the detection solution if the illumination of the mem-
brane is too strong. A good starting dilution is 1:1:8 of lumi-
nol/catalyst/H
2
O, but this requires initial testing.
32. Adding 1 volume of TCA stock to 4 volumes of protein sample
works more effi ciently than a precipitation using a fi nal con-
centration of 10 % TCA. However, the pellet requires more
methanol-acetone washes.
33. The samples should be blue. If the sample turns yellow, there
is residual TCA present, turning the sample acidic. This may
interfere with the electrophoretic separation. Adjust the sam-
ples by adding a few microliters of 1 M Tris pH 8.8 until the
color returns to blue.
34. Various different cell lines can undergo treatment with
refreshed media by mixing the spent medium (Pro293a) with
medium required by the selected cell line.
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