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protein fractions. For conducting normal immunoblots, prepare
extracts using buffer A.- For effi cient elution, ensure that the needle completely pierces
the microcentrifuge tube. The out pour of the liquid when
removing the needle is indicative of whether or not the tube
has been suffi ciently pierced. If elution is not complete, repeat
the centrifugation in step 9 for 15–20 s. - During the equilibration step, the 30 s fi ltration time may be
excessive depending on the vacuum strength. This time may be
adjusted accordingly. More importantly, monitor the fi ltration
to ensure that the wells do not dry out completely. Partial or
complete drying out of the membrane will affect binding of
proteins to the membrane. Once the buffer in a majority of the
wells has fi ltered through, turn off the vacuum and remove any
excess liquid from the remaining wells. This step will also be
indicative of the uniformity of the vacuum. - During fi ltration, ensure that there is no buildup of fl uid within
the tubing and around the fl ow valve to prevent backfl ow
which may ruin the experiment if not corrected quickly. This
can be prevented by increasing the vacuum strength by block-
ing the atmosphere line in the fl ow valve. We highly recom-
mend getting acquainted with the apparatus before proceeding
with the experiment. We usually see buildup around the fl ow
valve after assembling the apparatus due to excess retention of
buffer in the fi lter paper. In such instances, open the valve to
drain the buffer before loading the samples. - Sealing off the wells helps maintain uniform vacuum across the
apparatus and prevents drying. We fi nd that sometimes the fi l-
tration can be length depending on the amount of protein
used. In such instances keep adding buffer (few drops from a
P1000 pipet) to the sample wells that have fi ltered through to
prevent drying of the well until all samples have fi ltered
through. This should be done in addition to the wash step. - To avoid nonuniform destaining of the membrane, do not
pour the destain solution directly onto the membrane. Always
place membrane onto the solution. Monitor the bands on the
membrane to make sure that the higher dilutions (D3, D4) do
not destain completely. The incubation time in the destain
solution can be adjusted accordingly. - Keep all of the samples and buffers which are to be utilized on
ice throughout the procedure. - Incubating the cells with the FM stain for a longer duration of
time (1.5–2 h) may yield in better staining of the vacuoles.
However, this may be accompanied by increased background
staining. The concentration of the stain in culture may be
increased but we do recommend a minimum 1 h incubation
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