- Is the keratin identified by the protein ID service in my 1D gel band a contamination? Or is it a relevant human protein from the sample?
- How is it possible that only Keratin was identified in the sample although the protein band was clearly visible?
- Does this mean that keratin was the only protein in the sample? Or is the identification of other proteins impossible when even small amounts of keratin are present?
- The size of my protein and band cut from gel was 50-55kDa and Keratin is 66kDa, so how did the Keratin get into sample?
- Is there even a smallest possibility that something happened in your lab?
These are just few things that come to my mind as I’m thinking what might have gone wrong. It would be nice if you could help me with these concerns, before I’ll start the whole process once again.
Keratin identified in a gel band might be a real protein purified from the cells. But more frequently it is a contamination that occurred somewhere during protein purification and sample processing. Keratin is present in all dust in the lab from dead skin cells from humans and small pieces from your woollen sweater!
If the keratin contamination occurs prior to the gel electrophoresis, we observe it as protein bands in the gel around 55 kDa and 65 kDa. When identifying this keratin we see a high score and good sequence coverage, and often identify several keratin types. Since your gel band was observed at Mw 55 kDa, it is either a contamination that happened before the electrophoresis, or it was purified from the cells.
Keratin contamination can also occur after the electrophoresis during gel staining, or during gel scanning and spot excision if dust comes in contact with the sample. In such cases, the keratin amount is usually lower and you only observe a few keratin peptides in the mass spectra. It is still possible to identify the main protein component in the gel band.
At Alphalyse, we take extreme care to work in a clean dust-free environment. We handle all samples in 96-well plates together with quality control standards. Contamination from our lab is almost never observed.
The best advice to avoid keratin contamination is avoiding dust in the lab in general, and to always work with gloves. You should clean the electrophoresis and staining equipment and keep them free of dust. Also remember to always close the lids on pipette tips.
Below you find references to common protein and keratin contaminants observed in proteomics experiments:
The Common Repository of Adventitious Proteins, cRAP, is a list of proteins commonly found in proteomics experiments that are present either by accident or through contamination of protein samples.
This list of protein contaminants were observed in publication:
Large-Scale Proteomic Analysis of the Human Spliceosome. Juri Rappsilber, Ursula Ryder, Angus I. Lamond, and Matthias Mann. Genome Res. 2002. 12: 1231-1245 https://genome.cshlp.org/content/12/8/1231.full