The PCR conditions were as follows: 1 cycle at 94°C for 5 min; 35 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and 1 cycle at 72°C for 10 min. PCR products mixed with loading buffer were heated at 95°C for 5 min and quickly chilled on ice.
Samples (50 µg protein in 30-µl Laemmli buffer) were heated at 95°C for 5 min, resolved by 10% SDS-PAGE (50 µg/lane), and electro-transferred to PVDF.
Cell lysates in SDS-PAGE sample buffer were heated at 95°C for 5 min, separated by 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane.
Mouse or human protein samples in SDS sample buffer were heated to 95 °C for 10 min and separated on SDS-polyacrylamide gels.
The protein/DNA complexes extracted with elution buffer were heated to 65 °C for 6 h to reverse cross-links, then digested with proteinase K. DNA fragments were amplified in PCR with the ChIP assay primers containing the heat shock element sites in human Hsp70 promoter.
For antigen retrieval, slides in citrate buffer were heated in a microwave for 20 minutes and allowed to cool before blocking with 10% normal goat serum.
Aliquots of 50 micrograms in Laemmli buffer were heated in boiling water for 5 min, electrophoresed on 10% polyacrylamide gels, and transferred to PVDF membranes (Biorad, Hercules, CA).
Equal amounts of proteins mixed with Laemmli buffer were heated up to 95°C for 5 min and run on sodium dodecyl sulphate 10%-polyacrylamide gels before blotting on polyvinyldifluoride (PVDF) membranes (WESTRAN® clear signal; GE Healthcare Europe GmbH, Freiburg, Germany).
Proteins from the lysates were solubilized in 4× SDS loading buffer and were heated for 5 min at 100°C.
Raji, B95-8, C666, and SUNE2 cells were harvested and lysed in lysis buffer and were heated for 10 min at 98°C.
