I need a protocol that extracts both, whey proteins and caseins, to evaluate the protein profile of proteins present in dehydrated bovine milk by polyacrylamide gel electrophoresis (SDS-PAGE).
I do analysis of whey protein and caseins, but use LC-MS instead.
But a urea (6M) Sodiumcitrate (0,1 M) and 20 mM DTE work for me, and I am quite convinsed that this method are from SDS-PAGE. I use 200 µl milk to 1 ml buffer. Shake at 37°C in 1 hour, centrifuge and analyse the supernatant. Else I would say there is a huge amount of paper regarding this.
Thanks for the answer Lene Buhelt Johansen. So, I prepare the buffer with: urea (6M), Sodium citrate (0.1 M) and 20 mM DTE? Can I use DTT (dithiothreitol) as a reducing agent, instead than DTE? It is not necessary prior centrifugation, before the addition of the buffer?
Dear Cristine , I am working on the analysis of milk proteins using SDS-PAGE for more than 15 years and to evaluate the effect of dehydration on whole milk proteins you do not need to extract proteins , you can dissolve milk in water (13 %)and analyzed in SDS-PAGE directly.
But if you need to extract total milk proteins ( caseins and whey proteins) you can do that by treating liquid milk with 24% TCA (1:1).
If you need any more informations or references regarding this subject please inform me.
Dear Jasim Al-saadi thank you for your help. I will try both techniques to evaluate the profile of the bands. If possible I would like to have access the references that you have of this subject. Once again, thank you so much!@
Lene Buhelt Johansen What was the time and speed used in the centrifugation? Once again, thank you !
16.000 xg 10 min @4°C Christiane Almeida hope that you will find it useful.
- Badges
- Science method