Analysis of Cellular Proteins

5x10^-2x10^ cells (1-2 10 cm dishes) were suspended into 10 mis of PBS using a rubber policeman, pelleted at 500 x g, resuspended in 10 mis of PBS, re-pelleted and stored at -70 °C. Cell pellets were resuspended in 100 pi of Hypotonic Buffer (20 mM Tris-HCl (pH 7.0), 10 mMKCl, 2 mM phenylmethysulfonyl fluoride (PMSF), 1 pg/ml pepstatin, 1 pg/ml leupeptin, 1 pg/ml aprotinin), insoluble materials pelleted at 100,000 x g for 1 hour and supernatants (soluble fractions) retained for storage at -70°C. Pellets were washed with 500 pi of Hypotonic Buffer, resupended in 100 pi CSK Buffer (10 mM Tris-HCl (pH 7.6), 1% (v/v) Triton X-100, 2 mM PMSF, 1 pg/ml pepstatin, 1 pg/ml leupeptin, 1 pg/ml aprotinin) and incubated at 4°C for 5 minutes. Insoluble materials were pelleted at 20,000 x g for 10 minutes and the supernatants (Triton soluble fraction) retained for storage at -70°C. Pellets were washed with 500 pi of CSK Buffer, resuspended by sonication in 100 pi of CSK buffer by sonication and stored at -70°C.

2.2.4.2 Determination of Protein Concentration.

Modified Bradford Method (319) (Bio-Rad)

Aliquots of protein samples (2-10 pi) were diluted with ddH20 to 800 pi (in the case of insoluble protein samples 0.1% (w/v) SDS replaced ddH20). 200 pi of dye concentrate (Coomassie Brilliant Blue G-250 in phosphoric acid and methanol) was added to samples which were then incubated for 5 minutes. The absorbance of each sample (595 nm over a 1 cm path) was determined with a spectrophotometer (Beckman). BSA (Fraction V) (Sigma)

samples containing 0-20 mg/ml were used as standards.

2.2.4.3 SDS-Polyacrylamide Gel Electrophoresis.

(Biorad-Mini Protean II)

Cellular proteins were separated on 10 or 12%, 1.5 mm thick, vertical, discontinuous polyacrylamide/SDS gels. Separation gels containing 10 or 12% (w/v) acrylamide (30% stock acrylamide:bisacrylamide 37.5; 1), 0.375 M Tris-HCl (pH 8.8), 0.1% (w/v) SDS, 0.05% (w/v) Ammonium persulphate (APS), 0.05% (v/v) Temed were incubated for 30-60 minutes to allow to polymerize. Gels were overlayed with stacking gels of 4%(w/v) acrylamide, 0.125 M Tris-HCl (pH 6.8), 0.1% (w/v) SDS, 0.05% (w/v) APS and 0.2% Temed with teflon combs forming the sample wells. Gels were assembled vertically for electrophoresis with upper (cathode) and lower (anode) reservoirs containing IX Running Buffer (0.025 M Tris, 0.2 M Glycine, 0.1% (w/v) SDS). Samples were mixed with an equal volume of 2X Sample Buffer (0.125 M Tris-HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) p- mercaptoethanol, 0.002% (w/v) Bromophenol Blue) and loaded into wells (maximum 50 pi). Proteins were separated at a constant voltage of 180 V for -45 minutes. Prestained markers (Sigma or Gibco BRL) were used as molecular weight standards.

2.2.4.4 Visualization of Proteins.

{Coomassie ' Stain: Gels were incubated in 0.1% (w/v) Coomassiae Brilliant Blue, 45% (v/v) methanol, 10% (v/v) acetic acid for 1 hour. Excess stain was removed with Destain Buffer (40% (v/v) methanol, 10% acetic acid).

Silver Stain: (Bio-Rad) Gels were 'fixed' in 40% (v/v) methanol, 10% (v/v) acetic acid for 30 minutes, and 10% (v/v) ethanol, 5% (v/v) acetic acid for 2x 15 minutes, incubated in Oxidiser for 3 minutes and washed with ddH20 till the removal of excess oxidiser. Proteins were visualized by incubating gels in Silver Reagent for 15 minutes, washing in ddH20 and incubating in Developer with 3 changes. Staining was stopped with 5% (v/v) acetic acid.

2.2.4.5 Metabolic Labelling of N lE-115 Cells. (320)

Nl E- l 15 cell lines were replated into 3 cm tissue culture dishes at 2 x 10^ cells per dish. After a 48 hour recovery period the media was changed and the cell incubated with

normal media or media supplemented with 5 mM sodium butyrate for a fijrther 21 hours. Cells were washed several times in Methionine Deficient DMEM (Sigma) and incubated with 1 ml of Methionine Deficient DMEM, 10% FCS, 2 mM L-Glutamine, 50 pCi p^S]Methionine (1000 Ci/mmol, 10 mCi/ml), and if required 5 mM sodium butyrate for a further 3 hours. The media was removed and cell debris pelleted at 20,000 x g for 20 minutes. [^^S]Methionine labelled proteins were separated by isoelectric focusing in tube gels (pH 5-7/3-10 (4:1) ampholytes) (Section 2.2.7.4) prior to separation by molecular mass on 10% SDS-PAGE gels. Gels were fixed in Destain buffer (Section 2.2.4.4) for 15 hours, glacial acetic acid 2 x 20 minutes, 20% (w/v) 2,5-diphenyloxazole (PPO) in glacial acetic acid for 80 minutes and ddH20 for 20 minutes. The gels were vacuum dried (Slab Drier Bio-Rad) and exposed to X- Omat film at -70 °C for 10-14 days.

2.2.4.6 Immunological Analysis.

(Bio-Rad Trans-Blot)

Following the separation of proteins on polyacrylamide gels, the gels were equilibrated in Bjerrum and Schafer-Nielsen Transfer Buffer (48 mM Tris, 39 mM glycine, 0.0375% (w/v) SDS, 20% (v/v) methanol) at 4°C for 15 minutes with one change. Nitrocellulose filters (0.45 pm pore) (Schleicher and Schuell) and six pieces of 3MM filter paper were cut to the same size as the gels (up to 4 gels were transferred at the same time) and saturated with Transfer Buffer. Stacks of 3x 3MM filters, nitrocellulose filter, gels and 3x 3MM filters were placed onto the platinum anode, air bubbles displaced and the steel cathode placed onto the stack. Proteins were transferred onto the nitrocellulose at a constant voltage (15 V) for 1 hour at 4°C. Nitrocellulose filters were allowed to air dry and stored at 20°C if required.

Filters were incubated in PBS, 5% (w/v) dried milk powder (Marvel) or 3% BSA (Fraction V) for 1 hour at 20°C or overnight at 4°C to block non-specific protein binding sites and with primary antibodies diluted in Incubation Buffer (PBS, 1% (w/v) milk powder or 1% BSA, 5 mis per filter) for 1 hour at 20°C or overnight at 4°C. After 5x 5 minute washes in PB ST (PBS, 0.1% (v/v) Tween 20) the filters were incubated with a 1:1000 dilution of peroxidase conjugated anti-immunoglobulin secondary antibodies (Dako) in Incubation Buffer for 1 hour at 20°C. After 5x 5 minute washes in PB ST, the filters were incubated with

ECL detection reagents (1:1 ratio of ECL reagents A and B, Amersham) for 1 minute, covered in Saran Wrap and exposed to Hyperfilm-ECL at 20°C for 15 seconds - 30 minutes.

2.2.4.7 Immunocytochemistry.

Glass chamber slides (Nunc) were incubated with poly-l-lysine (100 pi per 1 cm^ chamber, 100 pg/ml) for 5 minutes, washed twice with ddH20 and air dried overnight. Chambers were inoculated with 2000 cells and allowed to recover at 37°C, 5% CO2 for 24-

48 hours. Cells were fixed with 3.7% (w/v) formaldehyde in PBS for 20 minutes, rinsed with PBS and permeabilized in 1% (v/v) NP40 in PBS for 20 minutes. Non-specific protein binding sites were blocked by incubating slides in Blocking Buffer (PBS, 3% BSA) for 1 hour. Slides were incubated with primary antibodies (diluted in 500 pi of Blocking Buffer) at 37° C for 1 hour and excess antibody removed with 3x 5 minute washes in PBS. Slides were incubated with fluorescein conjugated anti-immunoglobulin antibodies (1:10 dilution; Dako) 1:10 and rhodamine conjugated phalloidin (Stock: 0.1 mg/ml in methanol stored at -20°C diluted 1:100; Sigma) in Blocking Buffer at 37°C for 2 hours and washed in PBS (3x 5 minutes). Slides were mounted with Mowiol (10% (w/v) Mowiol 4-88, 20% (v/v) glycerol, 0.1 M Tris- HCl (pH 8.5), 2.5% (w/v) l,4-diazobicyclo-[2.2.2]-octane (DABCO)) and fluorochromes visualised by epifluorescence microscopy with excitation and emission filters suitable for fluorescein and rhodamine (Leitz).