of chromosomal proteins in plant cells
protocols for immunolabelling (US: immunolabelling ) were used to
test antibodies to mostly human chromosomal proteins such as CENP-E,
acetyl-histone H3 and phospho-histone H3 in Arabidopsis plant cells
and cultures, where they label histone H3 and other histones, phosho-histone
H3, and acetylated histones. See the antimethylcytosine
antibody labelling page for using that antibody to methylated DNA.
The protocols for tubulin / microtubules are also given.
Futher details of chromosome
preparation and in situ hybridization protocols are found in our
book, Practical in situ Hybridization, 2000. T Schwarzacher and
P Heslop-Harrison, published by BIOS and available from Sigma Chemical
(for c. GBP£30, USD$50, JPY10,500 or other currencies) or from any
bookshop. This is the 'book' referred to below and familiarity/cross
references to these methods is assumed. If there is another addition,
this protocol will probably be included.
ml 1 M PIPES pH6.8
10 ml 100 mM MgSO4
10 ml 100 mM EGTA
g paraformaldehyde in 80 ml water, heat for 30 min at 60C, and add
2-3drops of 1 N NaOH or KOH to clear and make to 100ml. Adjust
pH to 7 with H2SO4 . Aliquot and freeze at
-20C or keep at 4C for use in 1-2 days (see book Protocol 8.1)
is different to book appendix 1 (PBS)
As book, protocol 5.3
100uM stock solution of Taxol (e.g. Sigma Paclitaxel) in DMSO (keep
at 4C but warm to room temperature and agitate before use as it
precipitates). Taxol stabilizes microtubules, and the DMSO both
dissolves the taxol and helps penetration of all fixative components
at 1ug/ul in KPBS as chromosome counter stain
0.1% sodium borohydride in
KPBS (freshly made immediately before use)
BSA block: 1% BSA Fraction
5, essentially IgG free, in KPBS optionally containing 01-0.2% Tween-20
(see book for discussion of BSA grades)
Antibody solution: 0.1% Acetylated
BSA in KPBS/0.1-0.2% Tween-20 with appropriate antibody, typically
1:200 to 1:1000 dilution.
Poly-L-lysine coated slides;
make your own (see book) or buy ready coated from Sigma or
For Microtubules: Fix
young Arabidopsis inflorescences (groups of 5-10 buds) or cell suspension
in MT fix (2ml MT buffer, 2ml PFA, 8ul Triton X-100, 8ul Taxol)
for 1hour at plant growth temperature.
For Chromatin Proteins:
Fix young inflorescences or cells in KPBS fix: 2ml PFA, 800 ul 10xKPBS,
1.2 ml water, 8 ul Triton X-100, 8ul Taxol (used here for the benefit
of the DMSO)
The aim is to get chromosomes well spread and free of cytoplasm.
Much advice is given in the book 'Practical in situ hybridization'
chapter 5, although without acid fixation (which would destroy proteins)
it is difficult to get good spreads.
For Microtubules: Macerate
material in 0.5% Triton X-100 with the ends of a glass or metal
rod, place on poly-L-lysine coated slide and squash under a coverslip,
aiming to get a monolayer of cells, still intact in their walls.
Check metaphase index by counterstaining with DAPI after removing
coverslip by freezing. Check cell walls by phase-contrast
microscopy. Alternatively, make a cell monolayer in water and let
dry down onto poly-L-lysine coated slide.
For Chromatin Proteins: Dissect
the material in a drop of 0.5% Triton X-100, and place tissue with
metaphases onto a poly-L-lysine coated slide. Squash under coverslip,
or macerate with a glass/metal rod before putting on coverslip.
Freeze slide and flick off coverslip. To look for chromosomes/metaphases,
add a drop of DAPI stain and examine under UV microscope.
It is probably best to not
allow preparations to dry. After removing coverslip by freezing,
place into cuvette of KPBS for up to 4 h. Mark area of preparation
by scratching (see book).
re-fix preparations in fresh paraformaldehyde (Protocol 8.1 in book,
stage 6 paraformaldehyde fixation only) and wash in KPBS.
is likely to be a problem (e.g. from cell walls or chloroplasts),
place 250 ul of 0.1% sodium borohydride solution onto the maked
area of each slide and cover with a plastic coverslip. Once it has
stopped fizzing (2-5 min), wash slides well through 4 changes of
preparation by placing 250 ul BSA block onto the marked area of
each slide, cover with a plastic cover slip (see book) and leave
at room temperature in a humid chamber for 30 min,
Shake off block and
add primary antibody solution (50-100 ul per slide). Cover with
plastic coverslip and place in humid chamber for 1 hr at 37C
or 16-40h at 4C - typically overnight at 4C.
Counterstaining and Mounting
four times in KPBS (optionally with 0.1-0.2% Tween-20), then follow
protocols as protocol 9.2 for detection using appropriate secondary
antibodies. Use 1% BSA in KPBS wityh 0.1% Tween as block, and make
up antibodies in 0.1% Acetylated BSA in KPBS/Tween.
Follow protocol 9.3 for countertaining
and mounting. www.le.ac.uk
Trude Schwarzacher and Pat Heslop-Harrison
this page is under the 'methods' sidebar.
e-mails: TS32(a)le.ac.uk and PHH4(a)le.ac.uk
University of Leicester homepage: June 2005