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- For stable clone selection with puromycin, sufficient amount
of antibiotic should be used to kill all the non-transfected
cells. A concentration range of 2–10 μg/mL is sufficient to
kill all the untransfected cells. For a new cell line, puromy-
cin titration should be determined first then the selection
could be carried out. - Antibodies are typically recognizing epitope, a small portion of
the protein of interest and this domain may reside within the
3D conformation of the protein. Thus, to enable access of the
antibody to this part of the protein, it is necessary to unfold the
protein. Use a loading buffer (2× Laemmli buffer) containing
the anionic detergent sodium dodecyl sulfate (SDS) to denature
the protein. Take equal amount of protein (30 μg) solution and
add an equal volume of 1× Laemmli buffer. Boil the mixture at
95–100 °C for 5 min to unfold or denature the proteins. - Transfer condition may vary from system to system. So, the
condition may need to be optimized. - The antibody should be diluted in blocking (0.05%) solution
according to the manufacturer’s recommendation ratio. Primary
antibody incubation can also be carried out at room tempera-
ture for 1–3 h depending on the quality of the antibody. - The secondary antibody can be diluted in 0.05% skim milk in
PBST according to the supplier recommended ratio.
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