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- Protein fragments produced as a result of caspase-mediated
cleavage of specifi c substrates are not always possible to detect
by SDS-PAGE using antibodies raised against the full-length
protein. In this case, processing of target proteins in apoptotic
samples may be indicated by a decrease of substrate quantity. - Both caspase activity buffers can be stored for several weeks
at 4 °C. - In addition to classical FMK inhibitors, some suppliers
also provide a new generation of caspase inhibitors. For e
xample, Q-VD-OPh (Quinolyl-Valyl- O -methylaspartyl-[2,6-
difl uorophenoxy]-methyl ketone) is a cell-permeable, irrevers-
ible, broad spectrum caspase inhibitor that displays less toxicity
and longer stability than the FMK inhibitors. - Normally, due to denaturing effects of sample buffer and boil-
ing, the addition of protease inhibitors is unnecessary. - Cells will lyse in the BCA reaction buffer and release protein
content. - Alternatively, shear the DNA either by sonication or by
repeated passage through a 23-gauge needle. - Repeated loading of samples after storage in −20 °C requires
1–2 min heating at 95 °C in order to eliminate SDS
precipitations. - Wrapped in damp paper towels, a polymerized SDS-PAGE gel
can be stored at 4 °C for up to 1 week. - Since several factors, including quality of antibodies and
efficiency of the ECL solution used will influence the che-
miluminescence signal in SDS-PAGE immunodetection,
the optimal protein amount to be loaded has to be determined
experimentally. - Trapping of air bubbles is easily avoided if assembly of the
sandwich is performed submerged in transfer buffer. - Transfer effi ciency, quality of separation, and equal loading can
be controlled by Ponceau S Staining. Using this method,
microgram quantities of transferred protein can be detected
with a clear background and red protein bands. This staining is
reversible and allows further immunological detection.
Ponceau S stain solution: Dissolve 0.033 g Ponceau S in 10 mL
of water, add 0.3 mL of glacial acetic acid and make to 30 mL
with water. Store the solution at room temp.
Procedure for total protein detection:
(a) Immerse the blotted membrane in Ponceau S staining
solution and stain for 1–3 min.
(b) After staining, rinse the membrane in water until a distinct
protein pattern develops.
Magnus Olsson and Boris Zhivotovskyhttp://www.ebook3000.com