4. Sonicate two times for 30 s on ice. We use a Bioruptor™
sonication system (amplitude 30 and 0.5 s pulse on ice)
(optional).
5. Centrifuge for 15 min at 14,000 Â g at 4
C, collect the
supernatant, and estimate the total protein concentration
using a Bradford assay. A concentration of 3–4 mg/mL is
expected.
6. Heat 20 μg of total proteins from the soluble extract mixed
with 2Â Laemmli buffer at 95
C for 5 min and centrifuge
samples at 10,000 Â g for 30 s to bring down the condensate
and remove insoluble debris.
7. Load the centrifuged sample on an SDS polyacrylamide gel,
electrophorese, and transfer proteins from the gel matrix to a
nitrocellulose or PVDF membrane using your favorite device.
8. Block the membrane for 1 h at room temperature or overnight
at 4
C using 3% w/v dry skimmed milk or BSA solution in
PBS, incubate the membrane with an appropriate dilution of a
primary antibody directed against the affinity tag or the tagged
subunit in the same buffer for 1 h at room temperature or
overnight at 4
C.
9. Wash the membrane three times in TBST for 5 min each,
incubate with the recommended dilution of conjugated secondary antibody in TBST at room temperature for 1 h and
develop the Western blot.
The Western blot analysis of protein lysates from the parental
and from a modified cell line where XPB and XPB-GFP are detected
with an anti-XPB antibody is shown in Fig. 6a. As expected in the
case of a homozygous modification, the 90 kDa wild-type XPB
protein (lane 1) is replaced by the 130 kDa XPB-GFP fusion (lane
2). A heterozygous modification would result in the detection of
both a 90 kDa and a 130 kDa protein.
3.5.3 Fluorescence
Microscopy
We typically select four positive clones and include the
non-modified cell line as control. The target protein being fused
to GFP, expression of a fluorescent fusion protein can be checked
using an epifluorescence inverted microscope. Live or fixed cell
imaging can be performed.
1. Seed glass-bottom dishes/plates with clones that have been
tested positive until 70–80% confluency is reached.
2. For live cell imaging, change the medium to a phenol red-free
medium and observe cells using an inverted epifluorescence
microscope with adapted filters (i.e., an FITC filter for GFP
detection). Maintain cells at 37
C in the incubation chamber
of the microscope. Record images with a 63Â/1.4 NA
54
Sylvain Geny et al.
sonication system (amplitude 30 and 0.5 s pulse on ice)
(optional).
5. Centrifuge for 15 min at 14,000 Â g at 4
C, collect the
supernatant, and estimate the total protein concentration
using a Bradford assay. A concentration of 3–4 mg/mL is
expected.
6. Heat 20 μg of total proteins from the soluble extract mixed
with 2Â Laemmli buffer at 95
C for 5 min and centrifuge
samples at 10,000 Â g for 30 s to bring down the condensate
and remove insoluble debris.
7. Load the centrifuged sample on an SDS polyacrylamide gel,
electrophorese, and transfer proteins from the gel matrix to a
nitrocellulose or PVDF membrane using your favorite device.
8. Block the membrane for 1 h at room temperature or overnight
at 4
C using 3% w/v dry skimmed milk or BSA solution in
PBS, incubate the membrane with an appropriate dilution of a
primary antibody directed against the affinity tag or the tagged
subunit in the same buffer for 1 h at room temperature or
overnight at 4
C.
9. Wash the membrane three times in TBST for 5 min each,
incubate with the recommended dilution of conjugated secondary antibody in TBST at room temperature for 1 h and
develop the Western blot.
The Western blot analysis of protein lysates from the parental
and from a modified cell line where XPB and XPB-GFP are detected
with an anti-XPB antibody is shown in Fig. 6a. As expected in the
case of a homozygous modification, the 90 kDa wild-type XPB
protein (lane 1) is replaced by the 130 kDa XPB-GFP fusion (lane
2). A heterozygous modification would result in the detection of
both a 90 kDa and a 130 kDa protein.
3.5.3 Fluorescence
Microscopy
We typically select four positive clones and include the
non-modified cell line as control. The target protein being fused
to GFP, expression of a fluorescent fusion protein can be checked
using an epifluorescence inverted microscope. Live or fixed cell
imaging can be performed.
1. Seed glass-bottom dishes/plates with clones that have been
tested positive until 70–80% confluency is reached.
2. For live cell imaging, change the medium to a phenol red-free
medium and observe cells using an inverted epifluorescence
microscope with adapted filters (i.e., an FITC filter for GFP
detection). Maintain cells at 37
C in the incubation chamber
of the microscope. Record images with a 63Â/1.4 NA
54
Sylvain Geny et al.