Recently I'm trying to use Sep-pack C18 1 cc Vac Cartridge (Waters: SKU: WAT054955) to desalt digested peptides. However, the recovery rate is usually 40-60%. What is the normal recovery rate?
This will depend on the peptide, sample prep method and time. Run it through again. Review the manual too as they have many general suggestions in how to use.
That seems low. I had good recovery using Sep-Pak C18 Light. I was able to get closer to 95% recovery.
William Letter Thanks for your answer! I did trypsin digestion at 47ºC for 18hrs, the protein:trypsin ratio is 50:1. I used 0.1% Formic Acid, 70% Acetonitrile as Elution buffer and 0.1% Formic Acid, 0.5% Acetonitrile as Wash buffer for the Sep-Pak. Are there recommendations for my protocol?
Karen Kay Woolum Thanks for your answer! Could you help me with my protocol?
1. Condition Sep Pak with 1mL Elution Buffer.
2. Wash column twice with 1mL Wash Buffer.
3. Add peptide sample, push through slowly
4. Wash column twice with 1mL Wash Buffer.
5. Elute Sep Pak with 1mL Elution Buffer
6. Dry samples in speed vacuum concentrator.
Elution Buffer (0.1% Formic Acid, 70% Acetonitrile)
Wash Buffer (0.1% Formic Acid, 0.5% Acetonitrile)
That looks like how I did mine. I had a 30 mg column, so it takes more washing; we did 3 ml of each wash. My peptide was a I125 on c(RGDyK). Any unreacted NaI was removed by Sep Pak. I had good success with water only and Ethanol only is wash and then elution. 1 ml each. Do you keep the wash buffer and check for your analyte in it? Maybe you are losing too much in the wash.
Wash multiple times. Experiment with different elution solutions. You may not be eluting the sample off the column. Elution solvent choice is very important. *This process is worked out through experiementation.
Zi Yang it is my pleasure to help fellow researchers. Especially students.
try to optimize your experiment by utilizing fairly slow loading and elution steps. The binding and elution kinetics of the peptides must be well-permitted during application, which dramatically affects recovery.
Secondly, what is your loading buffer composition after digestion? Did you neutralize the alkaline condition of the digest before the SPE procedure...
You may also try to switch FA with TFA which acts as an ion pair agent and improves the retention of the hydrophilic and relatively small peptides. Thus your recovery may increase.
Also, consider the polarity of the resulting peptides...if highly lipophilic peptides are the case you may increase the organic composition of the eluent. Acidified ACN should be OK, not a fan of other organic solvents for elution...
Also, verify your digestion was successful. With inefficient digestion, you just clog the SPE resin due to the limited proteolysis leading to larger biopolymer fragments
These are the things I may indicate as addition to the suggestions above...
İsmail Emir Akyildiz Thanks for your answer!
1. I did the wash, binding and elution with vacuum because I found the gravity didn't work, but the vacuum was pretty slow.
2. The loading buffer condition was 100mM TEAB with 0.5% FA, I did acidified my samples before SPE.
3. I will try to use TFA after!
4. I will try gradient ACN for elution buffer.
5. I didn't run a gel to check the digestion efficiency, but I did a Peptide BCA assay and confirmed a pretty good digestion.
Thank you again for the detailed suggestions!!
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