Cellular protein analysis

2.3.1 Preparation of whole cell extracts for SDS-PAGE

Reagents

2x gel sample buffer (GSB) contained lOOmM Tris-HCL pH6.8, 20%

glycerol, 0.2M DTT, 4% sodium dodecyl sulphate (SDS), 0.02% bromophenol blue. Aliquots of 2x GSB were stored at -20°C.

Cells were counted on a haemocytometer and re-suspended in 50pl of lx PBS per 106 cells. An equal volume of 2x GSB was then added. Cells were sonicated using a W0385 sonicator (Hearsystems-Ultrasonics Inc.) and, following sonication, samples were heated at 100°C for 5 minutes on a dry heating block.

2.3.2 Preparation of cytosolic and nuclear extracts for SDS- PAGE

Reagents

Low salt detergent lysis buffer contained lOmM HEPES pH7.9, 1.5mM MgCE, lOmM KC1 and 0.1% NP40 and was stored at room temperature.

High salt buffer contained 20mM HEPES pH7.9, 420mM NaCl, 1.5mM MgCh, 0.2mM EDTA and 25% glycerol and was stored at room temperature.

Nuclear and cytosolic extracts were prepared using a method previously described (Brennan and O’Neill, 1995). Extracts were prepared on ice.

Cytosolic extracts were prepared by lysis of cells for 5 minutes in lOOjul of low salt detergent lysis buffer per 107 cells. Low salt detergent lysis buffer was supplemented with ImM phenylmethoslfonyl fluoride (PMSF), a 1:100 dilution of Phosphatase Inhibitor Cocktail I (Sigma) and a 1:100 dilution of

Phosphatase Inhibitor Cocktail II (Sigma). These supplements were added immediately prior to use due to their short half-lives. Following centrifugation (13,000 rpm, 5 minutes, 4°C), supernatant was collected (cytosolic extract).

Nuclear extracts were prepared by incubating the pellet for 15 minutes in 60pl of high salt buffer per 10 cells. High salt buffer was supplemented with a 1:100 dilution of Phosphatase inhibitor cocktail I and a 1:100 dilution of Phosphatase inhibitor cocktail II immediately prior to use. Following centrifugation (13, OOOrpm, 5 minutes, 4°C), supernatant was collected (nuclear extract). For extracts prepared from equivalent cell numbers, 50pi of 2x GSB was added to both the cytosolic and nuclear extracts. Alternatively, the protein concentration of the cytosolic and nuclear extracts was determined by a protein determination assay (section 2.3.3), prior to addition of an equal volume of 2x GSB to the extracts. Extracts were heated at 100°C for 5 minutes on a dry heating block.

2.3.3 Protein determination assay

Protein concentration was determined using a method based on that of Bradford (Bradford, 1976). Protein concentration standards were generated using a 1 mg/ml BSA (bovine serum albumin; Sigma) solution. Doubling dilutions of the 1 mg/ml BSA solution were prepared in a flat-bottomed 96 well plate. For the generation of a protein standard curve, triplicate lOpl aliquots of each BSA dilution was used. Three wells containing lOpl distilled H²O were included for the generation of the standard curve. A fixed volume (2-5 pi) of the nuclear or cytosolic extract was pipetted into the 96 well plate.

Protein assay reagent (Biorad, 500-0006) was diluted 1 in 5 with distilled H²O and 200pi was added to each standard and extract. The plate was incubated at room temperature for 5 minutes. A microplate reader (Biorad, 170-6850) was used to read the absorbance of each well at 570nm. The protein concentration of each extract was determined by plotting the OD57onm of a protein standard curve on an Excel spreadsheet, from which the concentration of protein in each sample extract was calculated.

2.3.4 DNA affinity precipitation of nuclear proteins

The ability of various transcription factors to bind to consensus DNA sequences was investigated by a DNA affinity precipitation (DNA-AP) protocol. This method is a way of measuring protein-DNA binding.

Streptavidin-conjugated agarose beads precipitate biotinylated oligonucleotide sequences from nuclear extracts of target cells. DNA affinity precipitation experiments were performed by a modification of a method previously described (Beadling et al, 1996).

Reagents

Dilution buffer contained 50mM Tris-HCl pH8, 0.25mM EDTA, lOmM NaF and 10% v/v Glycerol and was prepared when required.

Tris-EDTA (TE) contained lOmM Tris-HCl pH8 and ImM EDTA and was stored at room temperature.

2.3.4.1 Generation of double stranded oligonucleotides

The forward and reverse oligonucleotide sequences used are summarised in table 2.3. The consensus binding sites are underlined. Oligonucleotides were purchased from MWG Biotech. The forward oligonucleotides were supplied with 5’-biotinylation, whereas the reverse complementary sequence was not modified with biotinylation. Double stranded oligonucleotides were prepared using a method previously described (Brennan & Athie-Morales, 2001).

Oligonucleotides were diluted to a concentration of lpg/pl in TE and equal volumes of the forward oligonucleotide and the complimentary sequence were mixed. Preparations were incubated for 10 minutes at 95°C in a water bath.

The water bath was switched off and preparations were allowed to cool to room temperature. This gave a lpg/pl stock solution. For working dilutions, preparations were diluted to O.lpg/pl with TE buffer. Stock solutions were stored at -20°C. Working dilutions were kept at 4°C.

Table 2.3 Oligonucleaotides used for DNA Affinity precipitation. Phosphatase inhibitor cocktail I (Sigma), a 1:200 dilution of Phosphatase inhibitor cocktail II (Sigma), 0.5mM PMSF, 0.5mM NaV04 and 5mM DTT.

Supplements were added immediately prior to use due to their short half-lives.

Nuclear proteins were subsequently incubated (4°C, 60 minutes, rotating) with lpg of 5’-biotinylated double stranded oligonucleotide and 30jnl of pre­

washed streptavidin-conjugated agarose beads (50% slurry in PBS; Sigma, s- 1638) to allow binding of proteins to DNA. lpg of non-specific (STAT1) non- biotinylated double stranded oligonucleotide was also included in the mixture to favour the binding of low affinity proteins to reduce non-specific binding of proteins to the 5’ biotinylated oligonucleotide. Oligonucleotide conjugated beads were collected by centrifugation (4°C, 6000rpm, 5 minutes) and complexes were washed three times using dilution buffer supplemented with 0.5mM PMSF, 40mM NaCl and 5mM DTT immediately prior to use. DNA binding proteins to be analysed by 1-dimensional SDS-PAGE were eluted from the DNA by the addition of 2x GSB and heating at 100°C for 5 minutes on a dry heating block. DNA binding proteins to be analysed by 2-dimentional

(2D)-electrophoresis were eluted by the addition of 2D sample buffer (7MUrea, 2M Thiourea, 2% w/v CHAPS) and incubation at room temperature for 1 hour.