So I was analyzing HSP70 in plants by Western blot. However the bands I got was at around 150 kDa instead of 78 kDa indicated by the product information. I realized I didn't heat the sample+laemlli so the bonds couln't have been reduced. That explained why the bands appeared wrongly. Can someone please let me know if I am right and if there is other possiblities for those bands? Thank you so much.
Yes, you are most likely right. You need to heat the sample+laemlli to strip disulfide bonds. The protein may carry a lot of disulfide bonds as well, thus making it appear heavier.
You can increase BME upto 5mM, and heat sample followed by the centrifuge before loading on the gel. Good luck.
heating and adding reducing agent will disrupt the interaction between your proteins. If you skip either of these steps, you are likely to recover multimers.
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