especially in the Golgi, results in resistance to Endo H digestion. Both PNG-ase F and Endo H treatments reduce the
apparent molecular weight of the glycoprotein, which will
change the migration pattern of the protein on SDS gels
(as in Fig. 2b).
5. Detection of the HBV surface antigens and SVPs by the Monolisa HBsAg ULTRA kit (Bio-Rad) is based on the reactivity of a
mixture of monoclonal antibodies that are highly dependent on
the conformation of the “a” determinant of the S domain.
Therefore, chimeric HBV proteins containing insertions of
foreign epitopes within this region may be less well recognized
and their quantification underestimated. In our laboratory we
use western blotting under denaturing conditions and antibodies against linear epitopes (preS1) to complete this analysis.
6. Autoclave sucrose solutions at 100
C for 20 min and store at
4
C. Higher temperature and increased heat exposure may result
in sucrose breakdown and sugar caramelization, which will turn
the solution yellow-brown. Filter-sterilization is not an option
for concentrated sucrose solutions as filters tend to clog.
7. Split TB in equal volumes and add TR and the plasmid DNA
solution to each half, then mix them before cell transfection.
This will prevent DNA being precipitated when in contact with
concentrated TR.
8. We have also run experiments using HEK293T cells transfected
with empty pCi vector as a control and found no difference in
reactivity of cellular background proteins against anti-HBV
antibodies when compared to nontransfected cells.
9. Samples subjected to PNG-ase F and Endo H digestions are
denatured to increase de-glycosylation efficiency, which sometimes results in precipitation of proteins and signal loss in
western blots. This can be prevented by reducing the amount
of total proteins in the reaction volume.
10. Comparing samples migrated on SDS-PAGE under nonreducing and reducing conditions will provide a first indication on the
ability of the HBV chimeric proteins to form disulfide-linked
oligomers, a prerequisite for SVPs assembly (as in Fig. 2a).
11. Current commercial antibodies against HBV-S are conformation dependent; therefore, to detect HBV chimeric antigens by
western blot, antibodies against linear epitopes are preferred.
Membrane incubation with primary antibodies for 1 h is usually
sufficient for good signals in western blot, when using monoclonal anti-preS1 antibodies. However, overnight incubation is
recommended when the antigen is more diluted in analysed
samples. No additional membrane washing is necessary.
12. HEK cells are easily detachable; therefore, any washing steps
must be performed with care. Pipet solutions on the flask wall
92
Mihaela-Olivia Dobrica et al.
apparent molecular weight of the glycoprotein, which will
change the migration pattern of the protein on SDS gels
(as in Fig. 2b).
5. Detection of the HBV surface antigens and SVPs by the Monolisa HBsAg ULTRA kit (Bio-Rad) is based on the reactivity of a
mixture of monoclonal antibodies that are highly dependent on
the conformation of the “a” determinant of the S domain.
Therefore, chimeric HBV proteins containing insertions of
foreign epitopes within this region may be less well recognized
and their quantification underestimated. In our laboratory we
use western blotting under denaturing conditions and antibodies against linear epitopes (preS1) to complete this analysis.
6. Autoclave sucrose solutions at 100
C for 20 min and store at
4
C. Higher temperature and increased heat exposure may result
in sucrose breakdown and sugar caramelization, which will turn
the solution yellow-brown. Filter-sterilization is not an option
for concentrated sucrose solutions as filters tend to clog.
7. Split TB in equal volumes and add TR and the plasmid DNA
solution to each half, then mix them before cell transfection.
This will prevent DNA being precipitated when in contact with
concentrated TR.
8. We have also run experiments using HEK293T cells transfected
with empty pCi vector as a control and found no difference in
reactivity of cellular background proteins against anti-HBV
antibodies when compared to nontransfected cells.
9. Samples subjected to PNG-ase F and Endo H digestions are
denatured to increase de-glycosylation efficiency, which sometimes results in precipitation of proteins and signal loss in
western blots. This can be prevented by reducing the amount
of total proteins in the reaction volume.
10. Comparing samples migrated on SDS-PAGE under nonreducing and reducing conditions will provide a first indication on the
ability of the HBV chimeric proteins to form disulfide-linked
oligomers, a prerequisite for SVPs assembly (as in Fig. 2a).
11. Current commercial antibodies against HBV-S are conformation dependent; therefore, to detect HBV chimeric antigens by
western blot, antibodies against linear epitopes are preferred.
Membrane incubation with primary antibodies for 1 h is usually
sufficient for good signals in western blot, when using monoclonal anti-preS1 antibodies. However, overnight incubation is
recommended when the antigen is more diluted in analysed
samples. No additional membrane washing is necessary.
12. HEK cells are easily detachable; therefore, any washing steps
must be performed with care. Pipet solutions on the flask wall
92
Mihaela-Olivia Dobrica et al.