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When performing western blots for protein detection using the classical Laemmli method, experimenters often encounter difficulties with the detection of transmembrane proteins involved in lipid or fatty acid metabolism. A crucial phase in sample preparation is heating the samples to 100 °C in a Laemmli sample buffer containing SDS before separation by polyacrylamide gel electrophoresis (PAGE). In the current study, the analysis of several proteins was performed following modifications of the heating step during sample preparation. Multiple samples of the human Jurkat cell line were prepared using commercial or homemade Laemmli sample buffer. Samples were subjected to incubation at different temperatures for varying periods of time prior to separation by SDS-PAGE, transfer onto PVDF membranes and detection with specific antibodies. In samples incubated at temperatures of 25 °C, 40 °C, 70 °C and 100 °C, detection of the transmembrane protein elongase of long chain fatty acids 5 (ELOVL5) significantly decreased with temperature to a near total absence of signal at 100 °C. Heating (100 °C) the samples even for 1 min resulted in significant loss of ELOVL5 band intensity that was associated with the appearance of higher molecular weight immunoreactive materials. Loss of ELOVL5 band intensity was also observed with heating of samples at 100 °C prepared from HepG2, HEK293, MCF-7 and SKRB cells. The robust induction of ELOVL5 in stimulated primary T cells was not detected when sample were heated. The detection of fatty acid-metabolizing enzymes stearoyl-CoA desaturase-1 and long-chain-fatty-acid-CoA ligases 3 and 4 showed bands with significantly less intensity after heating at 100 °C compared to samples prepared at room temperature. Heating samples at 100 °C did not affect the detection of transmembrane proteins ERBB2 and five-lipoxygenase activating protein, or the soluble 5-lipoxygenase protein. Overall, the number of transmembrane passes of a protein was not predictive of loss of band intensity after heating, however this study indicates that sample heating can drastically affect the ability to detect proteins following separation by SDS-PAGE. This has implications for any detection methods that follow SDS-PAGE. |
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