v468 Chapter 6 细胞内结肠腺瘤样息肉蛋白核定位以及细胞质检测

Detection of Cytoplasmic and Nuclear Localization of Adenomatous Polyposis Coli (APC) Protein in Cells
 

Methods in Molecular Biology v468. chapter 6

Abstract

The adenomatous polyposis coli (APC) tumour suppressor gene is mutated in the majority of colon cancers.APC is a multi-domain protein whose distribution at different subcellular locations correlates withunique cellular processes. Our laboratory has focused on the link between APC subcellular location andfunction, and has characterized pathways for the trafficking of APC both into and out of the nucleus.Antibody specificity is an important factor in the determination of APC localization, and in this chapterwe outline a strategy for the unambiguous detection of APC using a combination of biochemical andcell biology approaches.

Key words: APC , Western blotting , Subcellular localization , Colon cancer , Immunofluorescence microscopy , Antibodies , APC detection , Electrophoresis .

1. Introduction

Colorectal cancer is one of the most common malignancies inthe world. The identification of mutations in the adenomatouspolyposis coli (APC) gene in patients affected by familial adenomatouspolyposis coli (FAP) was critical in understanding themolecular pathogenesis of colon cancer (1 – 3) . More important,APC mutations are detectable in > 80% of all inherited and sporadiccolorectal cancer patients and are directly linked with theearly onset of colorectal cancer (2) . Most APC gene mutationsare detected within the central mutation cluster region (MCR),and generate truncated APC peptides that lack a C-terminus. The truncated APC peptides display altered function, including a reduction in their ability to associate with microtubules (MT),facilitate correct chromosome segregation, and promote β-catenindegradation (2 , 3) .

The APC gene encodes a large protein consisting of 2,843amino acids with a predicted molecular weight of ?300 kDa,which interacts with proteins involved in the Wnt signalling pathwayand in cytoskeletal organization (3) . APC can shuttle betweenthe nucleus and cytoplasm and participates in a variety of cellularfunctions (4) . For instance, APC is known to regulate β-cateninlocalization and turnover (1 – 3) . In addition, APC is a cytoskeletalregulator and accumulates at the ends of MT bundles near theplasma membrane, where APC may contribute to cell migration(5 – 7) . Endogenous APC has been detected at several MT-associatedlocations, including membrane protrusions, kinetochores(8 , 9) , the mitotic spindle (10) , and centrosomes (11) by immunofluorescencemicroscopy. The ability of APC to shuttle betweennucleus and cytoplasm (reviewed in ref. (4) ) implicates its movementinto the nucleus, however many recent studies examiningnuclear APC have lacked suitable controls. In this regard, thespecificity of antibodies used to detect APC has caused discrepanciesin reports relating to APC accumulation in the nucleus. In most cases, APC has been detected by cell staining andmicroscopy, but not confirmed using biochemical methods. Wehave shown, using cell fractionation and Western blot analysis,that endogenous forms of APC are predominantly cytoplasmic,although a fraction of APC is localized to the nucleus, and thatAPC nuclear export activity is not abolished by truncating cancermutations (12) . These techniques are outlined in this chapter.

2. Materials


2.1. Cell Culture

1. Human SW480 (APC truncated at amino acid 1,338), HT-29 (APC truncated at amino acids 853 and 1,555), andHCT 116 (full-length APC) colon carcinoma cells are culturedin Dulbecco’s Modified Eagle’s Medium (DMEM)supplemented with 10% fetal bovine serum under standardtissue culture conditions.

2. A solution of trypsin (0.25%) and ethylenediamine tetra aceticacid (EDTA) (1 mM) is prepared, aliquoted, and can bestored at 4°C for 1 month and –20°C for longer periods.

3. Leptomycin B (LMB): working solution is diluted in ethanolat 2 μg/μL stored in aliquots at –20°C. Treatment ofcells is for 5 h at a dose of 8 ng/mL.


2.2. RNA Interference to Silence the Expression of APC

1. LipofectAMINE 2000 from Life Technologies, Inc. (Invitrogen, Carlsbad, CA; Cat. No 11668-019) is supplied in liquidform at a concentration of 1 mg/mL. Store at 4°C. Do notfreeze.

2. Double-stranded 21-mer RNA oligonucleotides homologousto sequences in human APC are purchased as purifiedduplexes (e.g. Qiagen-Xeragon Inc., Valencia, CA). TheDNA target sequence is 5,867–AGGGGCAGCAACTGATGAAAA.


2.3. Cell Fractionation

1. Cytosolic and nuclear extracts are prepared using a fractionationNE-PER kit from Pierce biotechnology (Pierce, Rockford,IL; Cat. No 78833) according to the manufacturer’sinstructions.

2. Bio-Rad protein solution (5×): this is a commercial solutionfrom Bio-Rad Laboratories (Hercules, CA; Cat. No 500-0006) based on the Bradford dye-binding procedure, whichmeasures the color change of Coomassie Brilliant Blue G-250 dye when it binds to protein.


2.4. Sodium Dodecyl Sulfate (SDS)– Polyacrylamide Gel Electrophoresis (PAGE) for APC Truncated Forms

1. Laemmli Sample buffer (4×): 250 mM Tris-HCl, pH6.8, 8%(w/v) sodium dodecyl sulfate (SDS), 40% (w/v) glycerol,and 0.1% (w/v) bromophenol blue. Store at room temperatureand add β-mercaptoethanol to 560 mM before using.

The aliquot with β-mercaptoethanol should be discardedafter use. Diluted concentrations of this buffer can be preparedwhen the volume of the samples is small.

2. Running buffer (5×): 0.125 M Tris base, pH to 8.3 with HCl,0.96 M glycine, and 0.5% SDS. Store at room temperature.

3. Separating buffer (1×): 1.5 M Tris-HCl, pH 8.7, and 0.4%SDS. Store at room temperature.

4. Stacking buffer (1×): 0.5 M Tris-HCl pH 6.8, and 0.4%SDS. Store at room temperature.

5. Forty percent acrylamide/bis solution (29:1). This is a neurotoxinsolution and so care should be taken to avoid exposure. Use gloves and a mask to prepare this solution. Store at 4°C.

6. N , N , N , N ￠-Tetramethyl-ethylenediamine (TEMED). Storeat room temperature, but note that the quality declines afteropening the bottle.

7. Ammonium persulfate (APS): prepare 10% solution in waterand immediately freeze in single-use (500-μL) aliquots at–20°C. A working aliquot can be kept at 4°C for no morethan 1 month (see Note 1 ).

8. Isopropanol. Store at room temperature.

9. Pre-stained commercial molecular weight markers.

10. Gel electrophoresis apparatus.


2.5. Western Blotting for APC Truncated Forms

1. Blotting buffer (5×): 0.125 M Tris-HCl pH 8.3, and 0.95 Mglycine. Store at room temperature and cool before using. Toprepare 1× solution, add 20% (v/v) methanol (see Note 2 ).

2. Nitrocellulose membrane from Millipore (Billerica, MA) and3MM chromatography paper from Whatman (Maidstone, UK).

3. Red Ponceau solution (1×): 0.2% red Ponceau powder, 3% trichloroaceticacid (TCA) in water. Store at room temperature.


2.6. Agarose Gel Electrophoresis to Detect Full Length APC (~300 kDa)

1. Laemmli sample buffer (4×): 250 mM Tris-Cl, pH 6.8, 8% (w/v) SDS, 40% (w/v) glycerol, and 0.1% (w/v) bromophenolblue. Store at room temperature and add β-mercaptoethanol(to 560 mM) before using. The aliquot with β-mercaptoethanolshould be discarded. Diluted concentrations of this buffercan be prepared when the volume of the samples are small.

2. Laemmli running buffer (5×): 125 mM Tris base, 960 mMglycine, and 0.5% (w/v) SDS.

3. Tris buffer EDTA (TBE) (1×): 89 mM Tris base, 89 mMboric acid, and 2 mM EDTA, pH 8.0. Store at room temperature.

4. 2.5% Agarose I in (TBE)/0.1% SDS.

5. Pre-stained commercial molecular weight markers


2.7. Capillary Blotting for Full-Length APC

1. Tris-buffered saline (TBS) pH 7.6 (1×): 20 mM Tris-HCl,pH 7.6, and 137 mM NaCl. Store at room temperature.

2. Nitrocellulose membrane from Millipore and 3MM Chrchromatography paper (Whatman).


2.8. Detection of Full-Length and Truncated APC on Nitrocellulose Membrane

1. TBS pH 7.6 (10×): 200 mM Tris-HCl, pH 7.6, and 1.37 MNaCl. Store at room temperature.

2. TBS with Tween (TBS-T) (10×): prepare a TBS solution (X1)and add 0.1% (v/v) Tween-20.

3. 5% (w/v) Non-fat dry milk in TBS-T.

4. Primary antibody: APC (Ab-1) antibody from OncogeneResearch.

5. Secondary antibody: anti-mouse IgG conjugated to horseradishperoxidase.

6. Enhanced chemiluminescent (ECL) reagents from Amersham(GE Healthcare, Piscataway, NJ).

7. Hyperfilm ECL: High performance chemiluminescence film(Amersham).


2.9. Immunofluorescence Microscopy for Detection of Full- Length and Truncated APC

1. Microscope cover slips (22×22 mm No. 1).

2. Phosphate-buffered saline (PBS) (10×): 1.37 M NaCl, 27mM KCl, 100 mM Na 2 HPO 4, and 18 mM KH 2 PO 4 (adjustto pH 7.4 with HCl if necessary). Autoclave before storageat room temperature. To obtain a 1× solution, dilute 1 partwith 9 parts of water.

3. Fixation solution: 3.7% (v/v) molecular biology-grade formalin.

Prepare a solution in PBS for each experiment atroom temperature.

4. Permeabilization solution: 0.2% (v/v) Triton X-100 in PBS.

5. Blocking solution: 3% (w/v) albumin from bovine serum(BSA).

6. The first, second, and third antibody solution are preparedin blocking solution.

7. Second antibody: anti-mouse or anti-rabbit conjugated tobiotin from Dako Corp. (Glostrup, Denmark) diluted 1:500.

8. Third antibody: Texas Red conjugated to avidin from VectorLaboratories (Burlingame, CA) diluted 1:500.

9. Nuclear stain: 0.05 μg/mL of Hoechst 33258 is prepared inblocking solution.

10. Mounting medium: Vectashield (Vector Laboratories).



3. Methods

APC is a nuclear–cytoplasmic shuttling protein with diverse functions.However, the immunostaining of cells to detect nuclearlocalization of endogenous APC is prone to false positive resultsdue to cross-reactivity of antibodies with other nuclear proteins.To obtain reliable results, it is important to compare APC localizationusing more than one approach, and if using fluorescencemicroscopy it is very important to complement those experimentsby using cell fractionation and immunoblotting. The use of RNAinterference (RNAi) provides a means to control for specificity ofthe cell staining of APC. In this section, we outline a procedure forthe detection of full-length APC (using agarose gel electrophoresis)and truncated mutant APC (using SDS-PAGE) from colontumor cell lines. A method for the staining and imaging of APC infixed cells is also presented to complement the Western blotting,and some details are provided to incorporate the use of anti-APCsmall interfering RNA (siRNA) and of LMB (nuclear export inhibitor)to observe the specific silencing and nuclear sequestration ofAPC, respectively (illustrated later by appropriate data figures).


3.1. Cell Culture,Tranfection, and Preparation of Samples

1. The colon cancer cell lines are maintained in 75-cm 2 tissueflasks and upon reaching confluence are passaged usingtrypsin/EDTA. At 24 h before transfection, seed the experimentalcultures into 25-cm 2 flasks for Western blot analysis,or seed the cells onto sterile coverslips for analysis by immunofluorescencemicroscopy. One 25-cm 2 flask or one coverslipis required for each experimental data point.

2. For RNAi, transfect the cells when at 50% of confluence (seeNote 3 ) with 6 μg siRNA duplex per 25-cm 2 flask or 3 μgsiRNA per standard coverslip in 2 mL or 1 mL of DMEMmedium, respectively (without serum or antibiotics, seeNote 4 ). Transfection of RNA duplexes is performed usingLipofectAMINE reagent. Add the reagent to cell mediumand incubate for 6 h. At the end of incubation, remove themedium and replace with complete DMEM. Continue incubationfor 48 h.

3. To transfect cells with plasmid, seed the cells onto coverslips.Transfect the cells when at 75% of confluence (see Note 3 )with 1–2 μg of DNA in 1 mL of DMEM (without serum andantibiotics, see Note 4 ) using LipofectAMINE reagent for 6h. At this point, remove the medium and incubate the cells incomplete DMEM for 48 h. Treat the cells with 8 ng/mL LMB for 5 h and harvest thecells for cell fractionation.

4. To harvest and to obtain the cell fraction protein extracts, collectthe medium of each flask in a 15-mL tube. Rinse the adherentcells with 2 mL of PBS and then collect them using 0.3 mL oftrypsin/EDTA. Centrifuge the cellular suspension at 500× g for7 min and remove the supernatant. Rinse the pellet with 500μL of PBS and centrifuge at 500× g for 7 min. Again removethe supernatant and separate the cellular pellet into nuclearand cytoplasmic fractions using the NE-PER kit (as directed bymanufacturer). Take an aliquot of each sample to determine theconcentration of protein using Bradford solution.

5. When loading the gels, cytoplasmic and nuclear fractions areloaded in a way to reflect the proportional cellular proteincontents. Equivalent amounts of each cell fraction (3:1 cytoplasmicto nuclear) are used; e.g. 60 μg of cytoplasm and 20μg of nuclear extract.

6. Transfer the nuclear and cytoplasmic extracts to Eppendorftubes. Add 4× Laemmli sample buffer to the samples to bringthe buffer to 1×.

7. Close the tubes and heat them for 5 min at 95°C. After coolingon ice, pellet debris by centrifuging at 13,000× g for 5 min.The samples (supernatant) are now ready for separation onacrylamide or agarose.


3.2. SDS-PAGE for APC Truncated Forms

1. The instructions below are for the preparation of minigelsusing the BioRad Mini-Protean separation system. It is criticalthat the glass plates are cleaned with a detergent, rinsedextensively with distilled water, and dried without remainingresidue.

2. Prepare 20 mL of a 1.5-mm thick, 8% gel by mixing: 3.8 mLof 40% acrylamide (29:1), 7.5 mL of separating buffer, 100μL of 20% SDS, 100 μL of 10% APS, and 8.5 mL of water.

Finally, apply 20 μL of TEMED, and pour the gel, leavingspace for a stacking gel, and overlay with isopropanol. The gelshould polymerize in about 20 min.

3. Pour off the isopropanol and rinse the top of the gel withwater (see Note 5 ).

4. Prepare the stacking gel by mixing: 1.29 mL of 40% acrylamide(29:1), 1.25 mL stacking buffer, 50 μL of 20%SDS, 50 μL of 10% APS, and 7.4 mL water. Finally, apply10 μL of TEMED. Use about 1 mL of this stacking gelsolution to cover the top of the separating gel, and theninsert the comb. The upper gel stack should polymerizewithin 20 min.

5. Prepare the “running” buffer by diluting 200 mL of the 5×running buffer up to final volume of 1 L with water in a measuringcylinder. Cover with Parafilm and mix by inverting.

6. Once the stacking gel has set, carefully remove the comb and,using a pipette or syringe with needle, flush the wells cleanwith water.

7. Assemble each gel unit to the electrical chamber and add therunning buffer, ensuring that it covers the wells. Load 30 μLof sample into each well. Different volumes can be loadeddepending on the sizes of the combs. Include one well forpre-stained molecular weight markers.

8. Complete the assembly of the gel unit and connect to a powersupply. Initially set the voltage to 70 V to allow the samples tostack, and then, as they enter the separating gel, increase thevoltage to 100 V (see Note 6 ). The blue dye fronts can be runoff the gel if desired.


3.3. Western Blotting for APC Truncated Forms

1. The samples that have been separated by SDS-PAGE are thentransferred to nitrocellulose membranes electrophoretically.

2. Prepare the blotting buffer by combining 200 mL of the 5×blotting buffer with 200 mL of methanol and water up to finalvolume of 1 L in a measuring cylinder. Cover with Parafilmand invert to mix.

3. Prepare a tray with blotting buffer and submerge two pieces ofsupport foam (a component of the Bio-Rad Western transferassembly), four sheets of 3MM paper, and a sheet of the nitrocellulosecut just larger than the size of the separating gel.

4. Disconnect the BioRad gel unit (or the equivalent) fromthe power supply and disassemble. Remove the stacking gel(optional) and rinse the separating gel in blotting buffer for5 min. Take a transfer cassette and place on the top side (thatwhich connects to the negative current) (see Note 7 ): onepiece of support foam, two pieces of 3MM paper, the separatinggel, then the sheet of nitrocellulose on top of the gel andtwo sheets of 3MM. Ensure that no bubbles are trapped in theresulting sandwich. The second wet foam sheet is laid on topand the transfer cassette is closed.

5. The cassette is placed into the transfer tank with blottingbuffer, the lid put on the tank, and the power supply activated.

Transfers can be carried out at either 0.090 A overnight or at0.300 A for 2 h at 4°C.

6. Transfer the nuclear and cytoplasmic extracts to Eppendorftubes. Add 4x Laemmli sample buffer to the samples to bringthe buffer to 1x

7. Once the transfer is complete, take the cassette out of thetank and carefully disassemble, with the top sponge, sheets of3MM, and gel removed. Rinse the nitrocellulose membranewith TBS. The coloured molecular weight markers should beclearly visible on the membrane. Stain the membrane with redPonceau solution to detect the proteins.


3.4. Agarose Gel Electrophoresis to Detect Full-Length APC

Due to the large size of wild-type APC (?300 kDa), it is muchmore effectively visualized after separation by agarose gel electrophoresisrather than SDS-PAGE. Samples are separated on a 3%agarose gel made in TBE/0.1% SDS. The agarose gel is preparedusing a vertical polyacrylamide gel apparatus with 1-mm spacers.First, pour a 1-cm 15% acrylamide plug to prevent leakage. Thenpour the molten agarose while it is still warm. There is no stackinggel . The running buffer is Laemmli buffer. Run the gel at 70 Vuntil the proteins in the 40–60 kDa range have migrated off thebottom of the gel.


3.5. Capillary Blotting for Full-Length APC

Transfer the proteins overnight onto a nitrocellulose membraneby downward capillary transfer as outlined below usingTBS/0.04% SDS as the transfer buffer:

1. On a 2.5-cm stack of paper towels, place three pieces of gelsizedWhatman 3MM chromatography paper with the toppiece pre-soaked in transfer buffer.

2. Place a pre-soaked, gel-sized piece of nitrocellulose membraneon top of the Whatman papers.

3. Place the gel on top of the membrane and cover the exposedpapers with plastic wrap.

4. Place two gel-sized pieces of pre-soaked Whatman 3MMpaper on top of the gel.

5. On opposite sides of the blotting stack, place a tray containingtransfer buffer.

6. Lay two pre-soaked, long pieces of Whatman paper across thetop of the gel setup so that the ends are submerged in the trayscontaining transfer buffer.

7. Cover the entire setup with plastic wrap and allow the transferto proceed overnight.

8. After overnight transfer, mark the position of protein standardson the nitrocellulose membrane with a pencil and rinsethe membrane with TBS-T. The detection of proteins withred Ponceau solution is not possible here.


3.6. Detection of Full-Length and Truncated Forms of APC

1. Incubate the membrane for 1 h in 5% non-fat dry milk inTBS-T at room temperature on a rocking platform.

2. Incubate the membrane with 1 μg/mL of APC (Ab-1) in 5%non-fat dry milk in TBS-T for 1 h at room temperature orovernight at 4°C on a rocking platform.

3. Wash the membrane three times, for 15 min each, in TBS-T atroom temperature on a rocking platform.

4. Incubate the membrane with a secondary antibody freshlyprepared for each experiment at a 1:8,000-fold dilution in 5%non-fat dry milk in TBS-T, at room temperature for 1 h.

5. Wash the membrane three times, for 15 min each, in TBS-T atroom temperature on a rocking platform.

6. Develop the membrane using ECL chemiluminescent detectionregents according to manufacturer instructions. Briefly,during the final wash, equal aliquots of each portion of theECL reagent are warmed separately to room temperature andthe remaining steps are done in a darkened room under safelight conditions. Once the final wash is removed from theblot, the ECL reagents are mixed together and then immediatelyadded to the blot, which is placed between two pieces ofsealable plastic (opened along one edge and closed after theECL reagents are added). Then, rotate by hand for 1 min toensure even coverage of the blot.

7. Remove the blot from the ECL reagents and place it betweenthe leaves of an acetate sheet protector that has been cut tothe size of an X-ray film cassette. Expose the membrane tofilm for 5 min. Adjust subsequent exposure times (from 30 secuntil 24 h) as necessary (see Fig. 6.1 for example of completedexperiment).

8. The membranes can be re-probed with other antibodies as afractionation or loading control.

Fig. 6.1. Detection of APC by biochemical assay and its confirmation using RNAi. AEquivalent amounts of cytoplasmic or nuclear extracts from cells treated +/- siRNA(APC, or control BRCA1 siRNA [ con ]) were separated by SDS-PAGE and analysed byimmunoblot. Truncated APC (150 kDa) was detected with antibody M-APC. The samemembrane was reprobed for topoisomerase IIα ( topoII ; 170 kDa) as a fractionationcontrol. APC siRNA effectively reduced cytoplasmic and nuclear APC by 80% relativeto untransfected cells. B Nuclear ( N ) and cytoplasmic ( C ) cell extracts were preparedfrom SW480 (APC mut/mut ), HT29 (APC mut/mut ), and HCT116 (APC wt/wt ) colon cancer cell lines.The endogenous truncated forms of APC were separated by SDS-PAGE (Section 3.2 ),and full-length APC was separated by agarose gel electrophoresis (Section 3.4 ) priorto detection by Western blot using the M-APC antibody (1:4,000 dilution). (Reproduced from ref. (12) with permission from the Nature Publishing Group) .


3.7. Immunofluorescence Microscopy of APC

The following is a general protocol for staining fixed cells andmicroscopic detection of APC. Cells can be transfected with anti-APC RNAi duplexes (see Section 2 and ref. (12) ) to confirm thatnuclear or cytoplasmic staining observed is actually APC. Notethat it is best to confirm siRNA efficacy first by Western blotting.

1. Remove the medium from cells (untransfected controls or 48 hpost-transfection), and wash the coverslips three times with PBS.

2. Fix the cells with 3.7% formalin/PBS for 20 min at roomtemperature. The formalin is discarded (into a hazardouswaste container) and the samples washed twice PBS.

3. Permeabilize the cells by incubation in PBS/Triton X-100for 10 min at room temperature, and then rinse twice morewith PBS.

4. Block the samples by incubating in 3% BSA solution for 1 h.

5. Remove the blocking solution without washing.

6. Apply 150 μL of the diluted primary antibody (e.g. Ali-28;Upstate) in blocking solution for 1 h at room temperature.

7. Wash three times with PBS.

8. Apply 150 μL of the diluted secondary antibody conjugatedto biotin in blocking solution for 1 h at room temperature(see Note 8 ).

9. Wash three times with PBS.

10. Apply 150 μL of avidin–Texas Red plus Hoechst solution inblocking solution for 40 min at room temperature.

11. Wash three times with PBS.

12. The samples are then ready to be mounted. The coverslipis carefully inverted onto a drop of mounting medium on amicroscope slide. The slides are air-dried at room temperatureand nail varnish is used to seal the sample. The samplecan be viewed immediately after the varnish is dry, or can bestored in the dark at 4°C for long periods.

13. The slides are then examined with any standard fluorescencemicroscope system, and images collected using a CCD camera(see Fig. 6.2 ).

Fig. 6.2. Detection of endogenous APC in SW480 by immunofluorescence microscopy.A Full-length pAPC-YFP (12) was transfected into SW480 colon cancer cells. After 48h, ectopic YFP localisation of APC-YFP was compared using different antibodies againstAPC protein by immunostaining. Positive co-localisation of each antibody with APC-YFPexpression is indicated (+). B Cellular staining patterns observed with different APCantibodies ( Ab ). SW480 or HCT116 were fixed on glass coverslips in formalin and analyzedby immunofluorescence microscopy as outlined in Section 3.7 . (Reproduced fromref. (12) with permission from the Nature Publishing Group) .



4. Notes

1. APS is not stable, and needs to be of good quality to polymerize.

If the aliquot is not fresh, there will not be polymerizationor the polymerization will be irregular.

2. The presence of methanol is necessary to fix the proteins tothe membrane after transferring. Otherwise, the proteins willbe lost from the gel into the buffer.

3. Transfect cells with siRNA when cells are at low density at thetime of transfection to avoid cell overgrowth increasing the genesilencing. The optimal cell density should be determined empirically for each cell line, and kept constant for all experiments to ensurereproducibility. For transfection of plasmid DNA, a higher confluenceis recommended at the time of transfection.

4. The presence of serum may inhibit cationic lipid-mediatedtransfection. It is recommended that medium with reducedconcentrations of serum such as Opti-MEM from Invitrogen(Cat. No. 31985-062) is used to dilute LipofectAMINE andnucleic acids before complexing.

5. The gel interface should be straight before applying the stackinggel. This is accomplished by using solvents of differentdensity than the gel.

6. The different pH between the running and stacking gelsenhances concentration of the samples at the stacking gel. Thepresence of SDS allows the samples to be separated based ontheir molecular weights (denatured gels).

7. It is vitally important to ensure this orientation or the proteinswill be lost from the gel into the buffer rather than transferredto the nitrocellulose.

8. This step is included to amplify the signal but can also increasethe background. It is possible to avoid this step by incubatingwith a second antibody anti-mouse or anti-rabbit conjugatedto the fluorophore.



Acknowledgments

The authors thank the National Health and Medical ResearchCouncil of Australia and the Australian Research Council forfunding support of this research.


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