Initially when I ran sds-page of my protein sample It gave many bands (around 10 bands in a lane) but now when I did SDS of the same sample it gave fewer bands than before (3-4 bands). Why is it so? Moreover, it showed smeared bands. Is my sample degraded or is there any contamination?
If proteolysis of your protein sample is occurring, consider how you are storing it. Keep it frozen, preferably at -80 C or in liquid nitrogen. Also, during the purification, consider adding a protease inhibitor cocktail.
If proteolysis of your protein sample is occurring, consider how you are storing it. Keep it frozen, preferably at -80 C or in liquid nitrogen. Also, during the purification, consider adding a protease inhibitor cocktail.
Why don't you post a picture of your before/after gels? Proteolysis usually generates more, rather than less bands.
What's the nature of your protein sample? Do u use every time reducing agent in the sample buffer?
I have already added EDTA as protease inhibitor and stored the sample (plant protein) in freezer at 4 degree.May be there is some other reason. Thank you.
Only EDTA as proteinase inhibitor is not enough ... It will inhibit basicly metalloproteinases ... It is better to add some proteinase inhibitor cocktail. At least add some inhibitor of cysteine proteinases...
Add protease inhibitor while lysing your sample. Infact your protein sample have degraded. After lysis, u can make aliquots of the sample and store it at -80 degree Celsius. If u have to do SDS-PAGE and western blotting, infact you can add reducing sample buffer ( if you want to have reduced protein) and boiled it and store it at-20 degree celsius. This will prevent your sample degradation as all type of proteases would have been degraded try once it might help. Good luck and happy working.
You are correct your protein sample is degraded. To get good protein extraction you should make a buffer containing protease inhibitor cocktail ( many companies sell these), all extractions and samples should be done on ice. When you have assayed your protein I would also recommend you aliquot out your desired amount for your planned experiment series ie several 50ug aliquots of each sample made up to a standard volume with the same buffer. You can also add your sample buffer (reducing or non reducing) and heat 95oc for 5mins, then spin down (8000rpm 30 secs) and freeze at -20oc short term or -80 long term.
I am agree with Carolin Elizabeth Dunk.
Part of protein sample which you wiil be to use in experiments need to archive (stor -80 oC). It's help you, you could come back to start of experiment.
Agree with Caroline Elizabeth Dunk.
In our practice here, we will add protease inhibitor cocktail during protein extraction too. Aliquot the lysates and store any leftover of protein lysate in -80C. The best is use the protein lysates as soon as possible without doubt and moreover, always work on ice during extraction (no matter samples or reagents) and centrifuge in 4C as well.
Good luck on your work~~~ :)
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