Protein localization-GFP-Arabidopsis spp. - Particle Gun

Nobody nobody at hgmp.mrc.ac.uk
Thu Jan 16 13:15:18 EST 2003


Gene Expression and Protein Localization in Arabidopsis





  OVERVIEW

To a large degree, the function of a protein can be inferred from its 
cellular compartmentalization and its interactions with other 
cellular components. Therefore, information on the subcellular 
distribution of a protein is crucial to determine its overall role in 
the cell. The histological distribution of the mRNA transcript 
reflects another important aspect; this aspect, however, has been 
covered in the in situ hybridization laboratory and will not be 
discussed here.



Several options are available to probe the subcellular localization 
of a given protein. In a few cases, it has been possible to design a 
specific way to localize a protein directly while inside the cell, 
based on a unique functional property or physical characteristic of 
the protein (light absorption or fluorescence). In the majority of 
cases, however, some form of indirect detection will be necessary. 
Methods that should be applicable to almost any protein will be the 
focus in this lab.





There are two major strategies for indirect detection:





A. Detection of the native protein: A specific antibody or an 
equivalent high affinity probe is used to label the target protein in 
situ. The probe is either directly labelled with a fluorescent tag or 
detected with secondary antibodies. Final observation is by either 
electron microscopy or light microscopy. While electron miscroscopy 
has higher resolution, it is also time-consuming and not very 
sensitive. In many cases, the light microscope can offer sufficient 
spatial resolution (Colasanti et al., 1993; Neuhaus et al., 1993; 
Wick, 1993; Matsui et al., 1995).





B. Detection of a recombinant version: Alternatively, a tag can be 
incorporated into the target protein and the modified protein 
introduced back into the cell by recombinant DNA technology. Thus, 
the fusion protein can be localized by monitoring the tag. There are 
three types of commonly used tags.



=46irst, epitope tags (8-11 amino acid stretches of well-characterized 
foreign proteins such as c-myc or hemagglutinin). These tags are 
small, thus minimizing the chance of altering the gross conformation 
(or subcellular localization) of the targeted protein. Monitoring the 
tag can be carried out according to standard immunological procedures 
with commercially available monoclonal antibodies specific for the 
chosen tag. The obvious advantage here is elimination of the need to 
generate specific antibodies for each new target protein.



Second, =FE-glucuronidase (GUS) acts as a reliable reporter enzyme in 
fusion proteins, especially for nuclear versus non-nuclear 
localization (Restrepo et al., 1990; Varagona et al., 1991 and 1992; 
Abel and Theologis, 1994; Abel et al., 1994; Citovsky et al., 1994; 
von Arnim and Deng, 1994). Although the in situ staining for GUS 
activity is easier than immunodetection, there are some potential 
drawbacks with this method. For example, the in situ enzyme assay can 
lead to a loss of spatial resolution.



Third, fluorescent proteins (e.g. GFPs) combine the advantage of high 
resolution with detection in vivo (Chalfie et al., 1994, Heim et al., 
1994; Prasher, 1995). This approach has been succesful in yeast and 
mammalian cells (e.g. Olson et al., 1995) and in Drosophila (Wang and 
Hazelrigg, 1994; Kerrebrock et al., 1995), and has also been 
developed for plants.



While a tag can obviously improve the specificity of detection and 
avoid the need for generating antibodies for every new protein to 
study, it can potentially alter the subcellular localization of the 
fused protein due to structural constraints introduced by the tag and 
by an aberrant expression level. However, genetic or biochemical 
experiments may clarify whether the fusion protein remains 
functional. Ideally, conclusions using one method should be 
corroborated by an independent approach.





AIM





During the two days allocated to this section, we will provide a 
theoretical overview and an experimental entryway into methods for 
transient gene expression and subcellular localization in 
Arabidopsis. The main course of events will be dominated by 
Experiment 1, immunofluorescent detection of proteins in protoplasts. 
In parallel, we will demonstrate the subcellular detection of 
proteins using GUS as a fusion marker (Experiment 2). Experiment 3 
will demonstrate the power of GFP as a subcellular marker and the 
transient expression of proteins using the particle gun.





Experiment 1: Immunofluorescence detection in Arabidopsis protoplasts



Experiment 2: Subcellular localization of GUS-fusion proteins in



Arabidopsis seedlings



Experiment 3: Transient gene expression of GUS and GFP by particle



bombardment







SCHEDULE





Experiment 1 meanwhile...





Day 1



1. Digest tissue 1) 09.00--11.00



2. Wash protoplasts 11.00 --12.00



3. Settle cells onto slides 12.00 --15.00 lunch, instruction set up 
GUS-staining



(Expt.2),



particle gun (Expt.3)



4. Fixation etc. of cells 15.00 --17.00 (leave over night)



5. Analyse GUS-stained



seedlings (Expt.2) 17.00--18.30





Day 2



5. Rehydration and block 1) 09.00--11.30 (TA)



6. Add first antibody 11.30 --13.00 lunch



7. Wash 13.00 --13.30



8. Add second antibody 13.30 --14.30 inspect cells from



transient assay



(particle gun) 2)



9. Wash 14.30 --15.00



10. Mount slides 15.00 --15.30



11. Analyse results 15.30 --18.30





1) This step will be set up by the TAs



2) Tissue transformed with the particle gun (Day 1) has been mounted 
for microscopy by TAs.





Experiment #1: Immunofluorescence detection in Arabidopsis protoplasts

INTRODUCTION



We will concentrate on the direct detection of a protein using 
specific antibodies. The major problem is to make the antigen 
accessible to the antibody, which is effectively blocked by cell 
walls and the cuticle. The effect of cell walls can be overcome by:





- Digestion of cell walls in a whole mount specimen



- Digestion of cell walls under isolation of protoplasts



- Sectioning





Several key factors need to be considered when choosing between these option=
s:



=46irst, autofluorescence in plant specimens can be a serious problem. 
Sometimes it is possible to work with tissues that show little 
autofluorescence, for example roots, which lend themselves to the 
whole-mount approach, but this is often impractical. More commonly, 
it is necessary to take additional measures to reduce 
autofluorescence, for example by washing out the interfering 
substances before microscopy. This is more easily achieved in single 
cells, such as protoplasts, than in whole mounts. In addition, 
careful selection of fluorophores and optical filters is necessary to 
reduce the interference of autofluorescence.



Second, the specimen has to be fixed to preserve the subcellular 
structures during the antibody incubation and wash steps. The 
trade-off: Gentle fixation will preserve antigenicity and keep 
autofluorescence low, but may not preserve the subcellular structures 
sufficiently. Harsh fixation conditions (especially glutaraldehyde) 
will increase autofluorescence.



Third, antigenicity of the target should be preserved. Strong 
fixatives will reduce antigenicity and may also unmask fortuitous 
epitopes. Embedding of tissue, associated with extreme changes 
between aqueous and non-aqueous media and exposure to a wide range of 
temperatures, also runs counter the preservation of antigenic sites.



Overall, while detection of a highly abundant antigen may be 
straightforward even after rough treatment of the tissue, only the 
very optimum of conditions may allow detection of a protein of low 
abundance.





The following strategy has allowed quite consistent detection of a 
wide variety of antigens in our hands.





1. Isolation of protoplasts from the tissue of choice



2. Binding of protoplasts to gelatin-coated slides



3. Fixation and permeabilization of the cells



4. Antibody incubations and washes



5. Mounting and inspection





This method has the following advantages:



(1) Access of the antibodies is very rapid.



(2) Fixation is quick and uniform.



(3) Autofluorescence is kept at a low level.



(4) The whole experiment can be carried out in one (long) day.





Disadvantages to be kept in mind are:



(1) Protoplasting may alter certain cellular structures.



(2) Analysis is confined to cell types amenable to protoplasting 
(mesophyll cells, etc.).







PROCEDURES





Part A: Isolation of Arabidopsis seedling protoplasts





1. Harvest one plate of seedlings, grown on solidified agar (GM 
medium) for 7 to 10 days.



-> Ecotypes Columbia and No-O work well. Using a small pair of curved 
scissors, cut the seedlings just above the agar surface to separate 
the roots from the rest of the seedling. Transfer the green organs to 
10 ml enzyme solution in a 125 ml Erlenmeyer flask or to 5 ml enzyme 
solution in a 6 cm diameter glass petri dish.





2. Apply vacuum for 1 minute in a desiccator/bell jar.





3. Shake slowly (40-70 rpm) for 90 to 120 minutes.





4. Shake for 10 minutes at 80 to 100 rpm to release the protoplasts.





5. Filter the cell suspension through a 75 micrometer nylon mesh into 
a 30 ml Corex glass tube.



-> Hold the folded mesh with a small plastic funnel or with one hand. 
Handle the cells gently with a pasteur pipette. Wash the flask or the 
dish with 5 ml of wash solution and pool this fraction with the first.





6. Spin for 2 minutes at room temperature at 150 g (1000 rpm) in a 
HB-6 swingout rotor with rubber adapters (up to 500 g is acceptable).





7. Pipette off and discard the 10 ml supernatant, resuspend 
protoplasts gently in 10 ml wash solution and incubate for 5 minutes.





8. Spin as above in step 6, remove supernatant, and resuspend the 
protoplasts in 0.5 ml wash solution. If necessary, the cells can be 
stored on ice.





Note: We thank Dr. Jen Sheen, Massachusetts General Hospital, Boston, 
MA, for communicating the basis of this protocol.







SOLUTIONS AND REAGENTS-1A





GM medium (1 liter)





sucrose 10 g



MS salts (Sigma M-5519) 4.3 g



MES buffer 0.5 g



1000x vitamins 1 ml





Adjust pH to 5.7 with KOH, add 8 g Bacto-Agar, autoclave in a 1 l 
bottle, cool to about 50=83C, and pour about 30 ml each into deep petri 
dishes (Nunc 4026).





1000x vitamins:



100 mg/ml myo-inositol



1 mg/ml thiamine



0.5 mg/ml nicotinic acid



0.5 mg/ml pyridoxine



Enzyme solution





cellulase R10 1%



macerozyme R10 0.25%



mannitol 0.4 M



MES 10 mM pH 5.7





Heat to 55=83C for 10 minutes to inactivate proteases and to accelerate 
enzyme solubilization, then cool to room temperature.





Add:



CaCl2 30 mM



=FE-mercaptoethanol 5 mM



BSA 0.1%





=46ilter through 0.45 micron filter. The solution can be stored for 
several months at -20=83C without apparent loss of activity.



Cellulase 'Onozuka' R-10 and Macerozyme R-10 are supplied by Yakult 
Honsha Inc. Co, Ltd., 1-1-19 Higashi Shinbashi Minato-ku; Tokyo; 105 
Japan.







Wash solution





mannitol 0.5 M



MES, pH 5.7 4 mM



KCl 2 mM





optional:



5 mM CaCl2, 2 mM malate, 2 mM ammonium sulfate, 1 mM potassium phosphate.





Part B: Immunofluorescence labeling of Arabidopsis protoplasts





1. Coat slides: Dip 8-well slides (Carlson Scientific, #100806) into 
a narrow beaker with coating solution, air dry for 30 minutes by 
leaning them against a support; rinse with water, then air dry and 
store at 4=83C in a microscope slide holder, wrapped in cling film.



-> Coated slides will be provided.





2. Place slides onto a pair of pasteur pipettes over wet paper towels 
in a dish with a tight lid (humid chamber).





3. With a pasteur pipette, apply a drop of protoplasts (ca. 50 =B5l in 
wash solution) to each of the 8 wells on the slide, cover the 
container and let the cells settle and adhere to the slide for 3 
hours at room temperature.



-> Make sure that the cells will stay moist throughout, if not stated 
otherwise.



-> Label slides with a pencil.



-> It is convenient to apply only one type of cells per slide and 
apply different antisera onto the different wells, rather than the 
other way round.





4. Gently remove most of the excess solution with a yellow tip from 
the wells of one slide. With a blue tip, immediately add 50 =B5l 
fixation solution to each well (need 400 =B5l per slide). Incubate for 
5 to 10 minutes at room temperature. Repeat for the other slides.



-> Lots of protoplasts don't stick to the slide and are lost at this step.



-> Work quickly to prevent cells from drying out while they are not 
protected by solution. Add and remove droplets of solution from the 
edge of the well, avoiding to touch the well itself.



-> Incubation times should be reproducible, but can be optimized for 
each antigen.





5. Permeabilize/destain the cells. Gently remove fixative solution 
with a yellow tip/Pipetman, again working from the edge of the well. 
Immediately add 25 =B5l permeabilization solution to each well and 
incubate for 5 to 10 minutes.



-> Keep the slide horizontal to make sure that this solution doesn't 
run off the slide.





6. Remove the permeabilization solution from one slide by aspiration 
or with a yellow tip/Pipetman. Immerse the slide into a glass slide 
holder/Coplin jar with acetone/ methanol (1:1 v/v, preferably kept at 
-20oC). Repeat for the other slides. Incubate slides for 10 minutes 
in the freezer (or on the bench). Using forceps, transfer the slides 
to a second Coplin jar with the same solution and incubate for 
another 5 minutes.



-> The slide holder is designed to hold up to 10 slides in back-to-back pair=
s.





7. Pull out the slides with forceps and air dry for at least 20 minutes.



-> At this stage all green pigment should be gone.



-> The slides can now be stored in a slide holder, wrapped in 
clingfilm, at 4oC over night.





8. Wash the slides once for 30 minutes with PBS (using the slide 
holder), then place them into the humid chamber.



-> Keep the samle moist in the humid chamber from now on.



-> It is most convenient and safe to remove solutions from the wells 
with a yellow tip at the end of a piece of tubing attached to an 
aspirator.





9. Apply 50 =B5l blocking solution per well (PHEM, 2% BSA); incubate 
for at least 30 minutes.





10. While the slides are being blocked, prepare the antibody 
solutions. About 10 =B5l per well is needed. Make a dilution in 
blocking solution in a 0.6 ml eppendorf tube. Spin the antiserum for 
10 minutes at 13,000 rpm to remove any insoluble aggregates of 
antibody and and transfer the supernatant to a new tube.



-> Leave the two left wells with blocking solution lacking antibody. 
These will serve as controls for autofluorescence and for the 
secondary antibody alone.



-> To reduce background fluorescence resulting from antibody 
aggregates, it helps to store (a 1 in 5 dilution of) the antibody 
stock in smalll aliquots at -80oC, after filtering it through a 0.2 
=B5m filter.



-> The concentration of antibody has to be determined empirically. A 
concentration of 5 =B5g per ml or a dilution of 1:100 is a point to 
begin with. Try 1:30 and 1:300 as well.





11. remove blocking solution by aspiration and apply 8=B5l of antibody 
per well. Incubate for 1 hour at room temperature or over night at 
4oC.



-> Make sure that the antibody cannot leak from one well to the next.



-> Overnight incubation tends to increase unspecific binding.





12. Wash. Remove first antibody solution, taking care that the sample 
doesn't dry out completely. Add 50=B5l PBS to each well and remove, 
then was with PBS containing 0.1% NP-40, then again with PBS (5 
minutes each).



-> The second and third wash are done by immersion of the slide into 
the slide holder.





13. While the slides are being incubated with the first antibody and 
washed, prepare the secondary antibody. Again, make 10 ml per well of 
a (suggested) 1:200 or 1:100 dilution in PBS, 0.5% BSA. Spin for 10 
minutes at 13,000 rpm and take only the supernatant to remove any 
insoluble aggregates. Apply 8 =B5l antibody to each well for 1 hour, 
making sure that it doesn't leak from one well to the next.



-> Incubate one of the two wells lacking first antibody with 
secondary antibody, and leave the other one with blocking solution 
alone (autofluorescence control).





14. Wash slides once with PBS, twice with PBS, 0.1% NP-40, and then 
once with PBS again, 5 minutes each.



-> The first wash is done on individual wells, the other washes in 
the slide holder.





15. Remove PBS by aspiration and quickly add about 2 to 4 =B5l mount 
solution to each well.



-> Spread out the mount solution with a yellow tip and add a 
coverslip. Add as little mount solution as possible, but try to avoid 
trapping air bubbles. To do this, initially add 2 =B5l; if this is not 
enough to cover the well, add aother 2 =B5l along the edge of the well. 
Seal the coverslip with nail polish and store slides in the dark.





16. Inspect slides at 1000x magnification (100x objective/lens) with 
immersion oil on a microscope with epifluorescence optics.



-> Beforehand, clean any gelatin from the back of the slide with 
water and a Kimwipe. Try to limit exposure of the samples to the 
microscope light, because this tends to produce a hazy background, 
rather than the more desirable black background. It is convenient to 
look for nicely fixed cells under the UV/blue channel (DAPI), then 
switch to the blue/green (fluorescein) or green/red channels (Texas 
Red, rhodamine). If desired, photograph with 400 ASA color slide film.



-> fluorescein bleaches out very easily, even in the anti-fade 
solution. Expose only briefly to epifluorescence light source.



SOLUTION AND REAGENTS-1B





Coating solution





Polylysine (reusable): 50 mg per liter in 10 mM Tris, pH 8.0; store at 4=83C=
=2E



Gelatin: Dissolve 1 g gelatin, 0.1 g chromic potassium sulfate 
(Chrome alum, Aldrich) in 100 ml warm water. Make this solution fresh 
each time.





PHEM (100 ml)



PIPES (MW 302.4) 60 mM 1.81 g



HEPES (MW 238.3) 25 mM 0.60 g



EGTA (MW 380.4) 10 mM 0.38 g



Mg2Cl (MW 203.3) 2 mM 40.6 mg



adjust pH to 6.9 with NaOH.





10x PBS (1 liter)



Na2HPO4 74 mM 10.6 g



NaH2PO4.H2O 14 mM 2.0 g



NaCl 1.5 M 90 g







=46ixation solution: 2% paraformaldehyde in PHEM.



-> To dissolve paraformaldehyde, heat gently to not more than 55=83C 
and vortex occasionally. The choice of fixative and the design of the 
fixation step are probably the most critical for the success or 
failure of the experiment.





Permeabilization solution: 0.5% NP-40 in PHEM





Acetone/methanol 1:1 (v/v)





Blocking solution: 2% BSA in PHEM





Mount solution: Antifade' (Molecular Probes, S-2828), freshly mixed 
to contain 1 mg/l DAPI (4',6-diamidino-2-phenylindole).



Experiment #2: Subcellular localization of GUS-fusion proteins in 
Arabidopsis seedlings





INTRODUCTION





GUS-fusion proteins have been employed extensively to ask whether a 
test protein can drive nuclear localization of the GUS enzyme marker 
(Restrepo et al., 1990). The fusion protein genes can be expressed by 
particle bombardment (Varagona et al., 1992), transformation of 
protoplasts (Abel et al., 1994) or from chromosomal transgenes 
(Varagona et al., 1991).



In this experiment, transgenic Arabidopsis seedlings carrying genes 
for GUS-COP1, GUS-NIa, or GUS will be provided.





PROCEDURES





Localization of Arabidopsis proteins with GUS in situ enzyme assay





1. For each sample, add 300 =B5l of stain solution to a well of a 
24-well tissue culture dish.





2. Using forceps, harvest seedlings grown on GM for 6 days and 
immediately place them into the stain solution.



-> Note: in general, a gentle prefixation of the tissue (2% 
formaldehyde in wash solution for 2 minutes), followed by washing, 
may preserve the subcellular localization better, but at the expense 
of the sensitivity of the detection. We did not notice significant 
differences in the relative GUS staining patterns with or without the 
fixation.





3. Incubate seedlings in stain solution at room temperature until the 
desired staining intensity is achieved.



-> Monitor progress under the stereomicroscope and/or:



-> Inspect under the light microscope in the presence of 1 =B5g/ml DAPI.





4. If desired, replace stain with wash solution.





5. Replace wash solution with fixation solution, incubate for 30'.



-> Wear gloves when handling fixation solution.





6. Replace fixation solution with 30% ethanol, leave on a rocking 
table for 10 minutes, then change to 50% and 70% ethanol. Incubate in 
70% ethanol overnight, gradually transfer back to wash solution and 
mount the seedlings in wash solution with 1 =B5g/ml DAPI on regular 
microscope slides. Seal the slides and store at 4=83C.





7. Observe under light microscope.





SOLUTIONS AND REAGENTS





Stain solution:





sodium phosphate, pH 7.0 100 mM



EDTA 1 mM



potassium ferrocyanide 5 mM



potassium ferricyanide 5 mM



Triton-X-100 1 %



X-Gluc 1 mg/ml



-> dissolve X-Gluc in a small volume of dimethylformamide by warming 
it for a few seconds at 50=83C.





Wash solution: 100 mM sodium phosphate, 1 mM EDTA, pH 7





=46ixation solution: 3.7% formaldehyde in wash solutionExperiment #3: 
Transient expression by particle bombardment





Particle bombardment has been used to introduce test plasmids into 
various tissues of Arabidopsis. Potential applications:





- Cell type specificity of promoter fragments



- Strength of promoter fragments in a defined cell type



- Subcellular localization of proteins





PROCEDURE





Particle bombardment





1. Prepare DNA:





=46or 5 shots @ 2 microgram plasmid DNA





- Place 50 =B5l of ethanol-washed tungsten particles into a 1.5 ml tube 
and vortex heavily.



-> Tungsten is prepared by suspension of commercially available 
tungsten powder at 50 mg per ml ethanol, thorough sonication, and 
washing with ethanol for four times. The particles are stored at 
-20=83C in 1 ml aliquots.



- Spin in microfuge for a few seconds and wash in 50 =B5l water.



- Repeat wash



- Resuspend in 50 =B5l water



- Combine:



50 =B5l tungsten particles in water



12 =B5l of DNA solution (1 mg/ml of a GUS-expression plasmid in water)



50 =B5l 2.5M CaCl2



20 =B5l 0.1M spermidine, free base





- Incubate 20 minutes on ice with occasional vortexing



- Add 200 =B5l ethanol, spin two minutes, and discard supernatant



- Wash particles 3 times in ethanol



- Resuspend particles in 30 =B5l ethanol, vortex well



- Pipette 5 =B5l of the particles onto each of five macrocarriers 
(flying disks (orange)).



-> Make sure the particles are well suspended, while dispensing them. 
Discard the last 5 =B5l (too lumpy).



-> Dry particles on macrocarrier on filter paper first in air then in 
a petri dish with drierite.





2. Harvest one plate of seedlings (ecotype No-O or Columbia), grown 
on solidified agar (GM medium) for 7 to 10 days. Use forceps to 
gently pull out the seedlings from the agar and arrange them 
(crowded) in the center of a petri dish of solidified MS medium with 
the roots in the center and the shoots on the periphery.





3. Bombardment with 1200 psi rupture discs, following specific 
instructions for BioRad particle gun.





4. After bombardment, seal the plates with parafilm and incubate over 
night under white light at 22=83C.





5. Stain the seedlings in a plastic dish with 3-5 ml of GUS-stain 
solution (see Experiment 2).



-> Monitor progress of staining under the stereomicroscope. To stop 
the staining, wash the seedlings in 100 mM phosphate, 1 mM EDTA (wash 
solution) and fix them in 3.7% formaldehyde in wash solution for 30 
minutes, then wash them again before mounting onto microscope slides.







Bombardment procedure





A. Setup





- Open pressurized helium tank, turning grey knob



- adjust pressure setting on helium tank gauge to 200 psi above the 
value of the rupture disk to be used.



- Switch on vacuum pump



- Switch on particle gun (left of three red buttons).





B. Shooting





1. Insert rupture disk (pressure disk, e.g. BioRad 165-2329): Unscrew 
grey disk holder from top of vacuum chamber, insert disk (can be 
sterilized in isopropanol beforehand), and screw grey disk holder 
back on.



-> Make sure disk makes a tight seal with holder and doesn't move 
while the holder is screwed back on.





2. Place macrocarrier (orange flying disk) with DNA side up into the 
small metal holder. Make edge of disk flush with holder by simply 
pressing the disk down with the red 'hat' supplied in the accessories 
box.





3. Unscrew lid (ca. 6cm diameter) from white Teflon plate. Insert 
stopping screen (BioRad 165-2336, package of 500) into bottom of the 
plate, then insert macrocarrier holder (with flying disk and DNA), 
DNA-side down, then screw lid back on. Place the whole set into the 
first shelf of the vacuum chamber with the tiny screw facing the 
door. -> The tiny screw can be loosened with an allen key in the 
accessories kit; then the position of the DNA can be adjusted up and 
down, if desired.





4. Place sample tissue into second shelf from the bottom. Close chamber door=
=2E





5. Pull vacuum (upper position on three-way middle switch of three 
red buttons).



-> I use 28 in Hg vacuum (pump will only deliver 27 in right now). 
Hold vacuum by quickly pressing three-way switch all the way down.





6. press fire switch (right button) continuously. Pressure will rise 
behind pressure disk, disk will burst, etc.





C. Open chamber, repeat procedure for second sample etc.





D. Shut down:



1. Close Helium tank (grey knob) .



2. Pull vacuum (about 15-20in) and hold.



3. Press fire switch to bleed helium line until pressure on gauge is 
close to zero.



4. Open pressure adjustment screw until handle runs freely



5. Bleed residual vacuum from chamber.



6. Shut off gun and vacuum pump.









SOLUTIONS AND REAGENTS





MS medium plates





4.3 g MS salts (Sigma), 1 mg thiamine, 10 mg myo-inositol, 180 mg 
potassium dihydrogenphosphate, 30 g sucrose, adjust pH to 5.7 with 
KOH, 1.2% bacto-agar; autoclave, and pour into shallow petri dishes.



-> optional: add 2.5 mg amphotericinB and 100 mg carbenicillin per liter.





Other solutions have been described in Experiment 2.



REFERENCES





Abel, S. and Theologis, A. (1994). Transient transformation of 
Arabidopsis leaf protoplasts: a versatile experimental system to 
study gene expression. Plant Journal 5, 421-427.





Abel, S., Oeller, P., and Theologis, A. (1994). Early auxin-induced 
genes encode short-lived nuclear proteins. Proc. Natl. Acad. Sci. 
(USA) 91, 326-330.





Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. 
(1994). Green fluorescent protein as a marker for gene expression. 
Science 263, 802-805.



-> GFP as a reporter for cell type specific promoter activity





Citovsky, V, Warnick, D, and Zambryski, P. (1994). Nuclear import of 
Agrobacterium VirD2 and VirE2 proteins in maize and tobacco. Proc. 
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Colasanti, J., Cho, S.-O., Wick, S., and Sundaresan, V. (1993). 
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and stomatal complex cells: association with predicted division 
sites. Plant Cell 5, 1101-1111.





Doonan, J., Zhang, H., and Traas, J. Indirect immunofluorescence on 
Arabidopsis seedlings. Arabidopsis - The Compleat guide.





Heim, R., Prasher, D.C., and Tsien, R.Y. (1994). Wavelength mutations 
and posttranslational autoxidation of green fluorescent protein. 
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Jefferson, R.A. (1987). Assaying chimeric genes in plants: The GUS 
gene fusion system. Plant Mol. Biol. Reporter 5, 387-405.





Kerrebrock, A.W., Moore, D.P., Wu, J.S., and Orr-Weaver, T.L. (1995). 
Mei-S332, a Drosophila protein required for sister-chromatid 
cohesion, can localize to meiotic centromere regions. Cell 83, 
247-256.





Matsui, M., Stoop, C.D., von Arnim, A.G., Wei, N., and Deng, X.-W. 
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Neuhaus, G., Bowler, C., Kern, R., and Chua, N.-H. (1993). 
Calcium/calmodulin-dependent and -independent phytochrome signal 
transduction pathways. Cell 73, 937-952.



-> This report describes a cryosectioning protocol, after stringent 
fixation of the tissue with formaldehyde, followed by detection with 
fluorescent antibodies. The antigens are abundant plastid proteins.





Olson, K.R., McIntosh, J.R., and Olmsted, J.B. (1995). Analysis of 
MAP4 function in living cells using green fluorescent protein (GFP) 
chimeras. J. Cell Biol. 130, 639-650.





Prasher, D.C. (1995). Using GFP to see the light. Trends Genet. 11, 320-323.



-> This review is accompanied by a series of others describing the 
application of GFP in different species.





Restrepo, M.A., Freed, D.D., and Carrington, J.C. (1990). Nuclear 
transport of plant potyviral proteins. Plant Cell 2, 987-998.



-> One of the first papers to describe the use of GUS-fusions for 
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von Arnim, A.G. and Deng, X.-W. (1994). Light inactivation of 
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