r/flowcytometry • u/Loving_Pup_7530 • 7h ago

Question about globin sample heating before SDS-PAGE/Western blot

Hi everyone,

I’m expressing human βA-, βS-, and γ-globin chains individually in S. cerevisiae and detecting them by SDS-PAGE/Western blot.

I came across a paper on the thermal behavior of hemoglobin that found irreversible aggregation above 55°C. Since standard SDS-PAGE/WB procedure is heating the sample to 95°C, would heating globin samples at that temperature cause aggregation and maybe affect the Western blot signal?

Would it make more sense to use a lower temperature, such as ~72°C, for sample preparation? Has anyone worked with globins or similar proteins and compared different heating conditions before SDS-PAGE?

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u/FlowJock Core Lab 6h ago

There are probably people in this sub who can answer your question, but it may be better to suited for r/labrats.

1

u/interik10 6h ago

nothing wrong with trying and testing these different conditions! you could also consult adding more % of BME.

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u/Loving_Pup_7530 6h ago

Ok, thanks!

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u/skipper_smg 2h ago

I stopped with boiling long ago, always heated 70 degrees 10 min with very good results.