I want to analyze the subunits in mitochondria complex . What I am doing is taking the certain density solution after sucrose gradient, and ten times diluted the sucrose solution containing the target complex with ddH2O. The diluted sample was add one third TCA to participate the protein, after 10 min on ice the tube was centrifuged at 14000g for 10min, and the pellet was washed with cold acetone for two times. The pellet was dissolved with water and the supernatant was used for MALDI analysis. While, I only got noise when I do the MALDI with this supernatent. Since there is always some pellet can not redissolved by water after TCA precipitation, which I think may cause sample loss. I tried another way to remove sucrose and salt and detergent in the sample. I used the Amicon filter column, 3K cutoff. I added 40 ul sample to the column, and add 400 water too. After mix the sample and water, the column tube was centrifuged at 14000g for 1hr30min. Then repeat the spin for three time with adding 450 water into the column tube each time to wash the sample. However, both of them did not show good MALDI signal. I only get some peaks lower than 14000 (m/z), or just noise. It is suppose to see more than 20 proteins in my sample, and the molecular weight range of the proteins should from 5K to 59K. The total protein concentration of the sample is measure as 1mg/ml. With this not very low concentration, I think my sample may have some inpurity that affect the protein crystallized with matrix.
Could anybody have suggestion with the sample clean for MALDI analysis? Thanks for any suggestion.
Dear Haiqing,
Is the purpose of your MALDI-TOF read out to see how many proteins you are recovering? Do you also need to ID these proteins?
Depending on what your experimental goal is will determine how best to recover and desalt proteins or peptides for MALDI-TOF MS.
Best,
Hediye
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH