To determine levels of the a-isoforms of the Na^-K^-ATPase pump and the SR Ca^^- ATPase pump, SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) was carried out according to the method of Laemmli (1970). The proteins were electrophoretically transferred to PVDF (polyvinyl difluoridine) membranes using the method described by Towbin (1979) and the PVDF membrane was probed using characterised antibodies to the proteins of interest (table 2.11).
Table 2.11. Composition of two-times SDS sample buffer, (w/v = weight/volume)
Compound Concentration
Tris-HCl (pH 6.8)* 100 mM
Dithiothreitol (DTT) 200 mM
Sodium dodecyl sulphate (SDS) 4% (w/v) (electrophoresis grade)
Bromophenol blue 0.02% (w/v)
Glycerol 20% (w/v)
*Tris-HCl buffer was made using Tris base (Trizma) and pH titrated to 6.8 with HCl
The infiltration of new cell types and collagen deposition in hypertrophied hearts could have a diluting effect on the protein of interest. Equal volumes o f sample were loaded from sham-operated and aortic constricted hearts. To correct for any differences in the amount of protein loaded from samples from different tissues, the samples were also analysed for calsequestrin, a protein that is not altered by the process of hypertrophy (Arai et a l, 1996; Tsutsui et a l, 1997; Shorofsky et a l, 1999; Schotten et a l, 1999;
Somura et al., 2001) and the results were normalised to calsequestrin levels as described by Meyer et al (1995).
2.10.1. Sample preparation
Samples of right or left ventricular myocardial tissue from sham-operated, aortic- constricted and debanded hearts were snap-frozen and stored in liquid nitrogen until used. Samples were prepared by adding 10 ml o f 50 mM tris(hydroxymethyl)aminomethane(Tris)-HCl buffer (pH 6.8) per gram of tissue; after which the sample was then homogenised on ice using a polytron tissue grinder. This made a 10% homogenate. The samples were divided into aliquots and stored at -70 °C until use.
A volume of the homogenate was thawed, then mixed with an equal volume o f two-times SDS sample buffer (Laemmli et al., 1970). The sample was not boiled as this may complicate the analysis o f membrane proteins such as Na^-K^-ATPase. Samples were centrifuged for 5 minutes at 20,000g and the supernatant applied to SDS-PAGE gels.
2.10.2. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) / Western blotting
Electrophoresis of the proteins from the prepared samples was carried out using the mini Protean II system (Bio-Rad Laboratories, Ca, USA) with 10% polyacrylamide gels (0.4 M Tris, 0.1% (w/v) SDS and 10% acrylamide, pH 8.8 with HCl) using a stacking gel (0.1 M Tris, 0.1% (w/v) SDS and 5% acrylamide, pH 6.8 with HCl). Equal volumes of sample (10 pi) were loaded on to the gel at the top of each lane using a pipette. Measurements with normal rat myocardium have shown that this gives about 30 pg of protein per well.
The negatively charged molecules o f sodium dodecyl sulphate (SDS) bind to the hydrophobic regions of the protein molecules, causing them to unfold into extended polypeptide chains. A reducing agent, dithiothreitol (DTT) was present in the sample buffer to reduce any disulphide bonds present within proteins. The individual proteins are thus rendered free from their associations with other proteins or lipid molecules and
rendered freely soluble in the detergent solution. The individual polypeptide chains migrate as a negatively charged SDS-protein complex through the porous gel of polyacrylamide. Under these conditions the speed of migration is greater the smaller the peptide, thus allowing separation o f the proteins according to molecular weight (Laemmli et a l, 1970).
After electrophoresis proteins were electrophoretically transferred to a PVDF membrane (Pharmacia AB, Stockholm, Sweden) with a semi-dry blotter (Pharmacia AB, Stockholm, Sweden) and a current of 100 mA per gel for 2 hours (Towbin et a l, 1979). The transfer buffer contained 20% methanol (v/w), 0.5 M glycine, 25 mM Tris and 0.01% SDS (w/v).
To reduce non-specific binding, PVDF membranes were blocked with 5% skimmed milk in phosphate-buffered saline (PBS) for at least 4 hours at room temperature. The membranes were then incubated with primary antibodies specific for the a-1, a-2 and a- 3 isoforms o f the Na^-K^-ATPase pump, sarcoplasmic reticulum Ca^^-ATPase (SERCA2) and calsequestrin (table 2.12).
2,10.3. Antibodies
Monoclonal antibodies and polyclonal antisera directed against the specific isoforms of the Na^-K^-ATPase pump and the SR Ca^^-ATPase pump were used (table 2.12). A mouse monoclonal antibody specific for ai-isoform was purchased from Upstate Biotechnology (USA). A monoclonal antibody specific for cli (McB2), raised against purified rat axolemma Na^-K^-ATPase, showing broad species cross-reactivity was a generous gift from KJ Sweadner (Massachusetts General Hospital, Boston MA, USA). A monoclonal antibody against mouse ag-isofbrm was purchased from Affinity Bioreagents Inc (USA). Polyclonal antisera against rabbit a2-isoform was purchased from Upstate Biotechnology (USA). A monoclonal antibody directed against mouse SR Ca^-ATPase (SERCA2A) and polyclonal antisera against rabbit calsequestrin were obtained from Affinity Bioreagents Inc (USA).
Table 2.12. Details o f the specific antibodies used to detect the a-isoforms of the Na^K^ ATPase pump, SERCA2 and calsequestrin
Antibody Iso form Source Reference
Mouse a l Upstate Biotechnology
(Monoclonal) (USA)
Rabbit a2 Upstate Biotechnology
(Polyclonal) (USA)
McB2 (rat) a2 KJ Sweadner Urayama et a l.
(Monoclonal) 1989
Mouse a3 Affinity Bioreagents
(Monoclonal) Inc (USA)
Mouse SERCA2A Affinity Bioreagents
(Monoclonal) Inc (USA)
Rabbit Calsequestrin Affinity Bioreagents
(Polyclonal) Inc (USA)
The tt], calsequestrin and SERCA 2 antibodies were incubated for 1 hour at room temperature. The a% and as antibodies were incubated overnight at 4 °C. Non-specific primary antibody binding was removed by washing the blots in PBS 0.05% Tween-20 with multiple changes o f wash buffer over 1 hour. Blots were then incubated with a secondary antibody, conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature (anti-rabbit IgG antibody (dilution 1: 1000) for a% and as and calsequestrin and anti-mouse IgG (dilution 1: 1000) for a j and SERCA2, Amersham UK). After both the primary and secondary antibodies were applied, the blots were washed again with multiple changes o f PBS 0.05% Tween-20 for I hour. The PVDF membrane was then soaked for approximately 1 minute in ECL (enhanced chemiluminescence) detection reagent (ECL system, Amersham, UK). This elicits a peroxidase-catalysed oxidation of luminol, and subsequently enhanced chemiluminescence where the HRP labeled protein is bound to the antigen on the membrane, thus enabling visualisation o f the primary antibody binding. The resulting light was detected by exposing the membrane to autoradiograph film (Hyperfilm ECL). The ECL images were digitised using a flatbed scanner (Hewlett Packard, ScanJet IIC ), and quantitative analysis performed using the NIH software (Freeware, NTH, Baltimore, USA).
After taking images, blotting integrity was confirmed by staining the PVDF membranes with 0.25% Coomassie brilliant blue (BDH, UK) in 10% acetic acid, 50% methanol for 5 minutes. The membranes were transferred to destaining solution (40% methanol, 7% glacial acetic acid) and agitated, to remove non-specific background staining.