Biomedical Engineering Reference
In-Depth Information
5. Fixation with PFA is the most widely used approach. However,
there are some antibodies which epitopes are destroyed by
PFA. Alternatively, 100 % methanol fi xation (either −20 °C or
RT) for 5 min can be used after PFA fi xation or as a fi xative
alone. In this case, use BSA blocking buffer (5 % BSA fraction
V in CMF-PBS) and BSA dilution buffer (1 % BSA fraction V
in CMF-PBS). Digitonin (50
g/ml digitonin in PBS) or
saponin (0.2 % in PBS) can be used instead of 0.2 % Tx-100.
100 % methanol at room temperature for 5 min also works well
to permeabilize after PFA fi xation for LC3 antibodies.
For antigens which have a cytosolic and membrane-
associated pool, it is worth considering a pre-permeabilization
step using very low concentrations of saponin (between 0.01 %
and 0.001 % in 80 mM pipes, 1 mM MgCl
2
, 5 mM EGTA,
pH 7.4) before PFA fi xation.
6. Primary antibodies from different species can be mixed
together in PBS-gelatin to save time. For instance, a common
mixture might be a rabbit polyclonal, a mouse monoclonal,
and a sheep polyclonal. The only caveat is the use of secondary
antibodies made in goat (e.g., goat anti-mouse) that will cross-
react with sheep primary antibodies. To avoid this, we use sec-
ondary antibodies raised in donkey. Both primary and
secondary antibodies should be used at the lowest possible
dilution. There should always be a control of secondary anti-
body/antibodies alone.
7. Quantifi cation is more robust if equal numbers of cells are
plated in each well as the samples are not normalized to pro-
tein content.
8. The investigators own treatment can be used here either in
parallel to amino acid starvation or as a stand-alone treatment.
Caution should be used when treating cells for prolonged
times (greater than 8 h) with BafA because of cytotoxicity.
9. It is best to process and run the samples ASAP; however, sam-
ples can be left in lysis buffer in the dish at −80 °C for a week
(put dish directly onto metal shelf in freezer).
10. Decrease 5× sample buffer if using gels with small well vol-
umes. It's important to load as much as possible (without
compromising sample solubilization) to maintain a good signal
to noise ratio.
11. Must use PVDF or PVDF-FL membranes with LI-COR.
Nitrocellulose membranes give a high background with LI-COR
but can be used for ECL.
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12. To obtain good data with MetaMorph, keep the boxes as small
as possible.
