Biology Reference
In-Depth Information
efficiency. In this paragraph, we will focus on different conditions of SDS buffer
elution.
The effective elution procedure also requires optimization for the specific pair of
proteins. Two factors have to be taken into consideration when the elution conditions
are optimized: (1) temperature and (2) presence of reducing agent. In our experi-
ments, most of the time, proteins are eluted at 37
C for 10 min in SDS sample buffer
(4% SDS; 50% glycerol; 0.2% bromophenol blue; 65 mM Tris/HCl pH 6.8) with
addition of 50 mM DTT.
In the experiment presented in
Fig. 17.3
, different elution temperatures and the
importance of DTT addition were tested. Protein denaturation of the lysate sample at
95
C resulted in detection of a smear at the top of the immunoblot, which represents
aggregated proteins. Denaturation at 37
C with addition of DTT allowed for
FIGURE 17.3
Comparison of different elution conditions. HEK293T cells were transiently transfected
with pHA-D
4.2
R. Immunoprecipitation with mouse anti-HA (16B12) was performed from total
cell lysate. Proteins were eluted from the beads at three different temperatures: 37, 50, and
95
C with or without addition of 50 mM DTT. After Western blot analysis, proteins were
visualized with mouse anti-HA (16B12) and antimouse IRDye 700 or rabbit anti-HA and
antirabbit IRDye 800. PM-mature, fully glycosylated plasma membrane receptor;
ER-ER-retained receptor.*Association of two heavy chains (2 50 kDa), **one heavy chain
(50 kDa), ***one light chain (25 kDa) of anti-HA sera.
