I purified the virus(H1N1/PR8) protein from the allantoic fluid by 20%-50% sucrose gradient, and I used the SDS-PAGE to separate the HA protein; I loaded 5ug protein per well; after coomassie blue stain, I couldn't see the protein, but in the western blot I used the anti HA antibody ,why I can see the multiple band.
Sample1 tube1-30%-40%
Sample2 tube1-20%-30%
Sample3 tube2-30%-40%
Sample4 tube2-20%-30%
Did you add reducing agent (BME or DTT) to your Sample buffer? Are you boiling the samples before running the gel? The protein appears to be in oligomeric states that are not being broken down by your current sample buffer conditions.
David Heisler Thank you for your reply. The attachment is the latest result, and I load BSA as a control to make sure BME is working. the sample I boil for 10 mins before running the gel. I still couldn't see the protein after coomassie blue stain.
The straightforward answer is that actual amount of HA protein content is too low to be picked up by coomassie stain, but is detectable by Western blot due to the enzymatic amplification of the detection reagent. Did you try silver staining the gel? That is much more sensitive than coomassie.
Not knowing how you determined your protein concentration for loading, I cannot speculate on what you are reading, but clearly, the HA protein is a fractional portion of the total.
Chang Yu Ko,
Your protein is oligomeric. Since reducing agents don't help to break them apart, the interactions do not involve cysteines. Although SDS breaks most non-covalent interactions, some are resistant to it. Is that a capsid protein? Also, you clearly see it on Coomassie, except it is smeared. Finally, I've never tried it myself, but people claim that Zn Statining is as sensititive as Silver, but faster, less expensive, and reversible:
https://conductscience.com/zinc-staining-protocol/
Good luck.
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