Keratin contaminations
Keratins are classic contaminants in proteomic studies. Their presence might have serious incidence on result quality. So as to avoid spots contamination, one must follow simple rules:
You : Keratins come from the skin, hairs and clothes
- Wear clean white coat and gloves all along the experiment
- Change gloves at each manipulation
- Avoid natural fibre clothes (wool, mohair…).
Material : Keratins come from dust
- Material in contact with polyacrylamide gels must be washed with detergents, rinsed in ultrapure water and dried with ethanol.
- Material must be kept away from dust
- When possible, manipulate in a laminar flow hood
Solutions : Solutions themselves may be a keratin source
- Polypropylene tube will be preferred
- Tips will be exclusively reserved to a proteomic use and prefer prepacked tips.
- Always wear gloves!
- Products of solutions will be exclusively reserved to a proteomic use.
- Buffers will be freshly prepared and stored at -20ºC.
Gel : “to have a maximum protein / acrylamide ratio”
- Whenever possible, prefer thin gel (0.75 mm)
- For SDS-PAGE, load empty lanes with Laemmli buffer to limit protein diffusion.
- Prefer colloidal blue staining procedure over silver staining. In case of silver staining, treat sample as soon sa possible.
- Store gels in acetic acid 1% at 4°C.
Precipitation
According to PMQ-PG-108-01
Acetone Precipitation
- Add 5 volumes of cold acetone to sample
- Vortex and incubate 30 min at -20°C
- Centrifuge 15 min at 20000 g
- Remove supernatant and let dry at room temperature
- Resuspend in appropriate buffer
TCA Precipitation
- Add TCA to a final concentration of 15% (w/v)
- Vortex and incubate 30 min at 4°C
- Centrifuge 15 min at 13000 g
- Remove supernatant
- Wash with cold acetone (~ 1 ml)
- Centrifuge 10 min at 13000 g
- Wash with cold acetone (~ 1ml)
- Centrifuge 10 min at 13000 g
- Remove supernatant and let dry at room temperature
- Resuspend in appropriate buffer
Ethanol Precipitation
- Add 2 volumes of cold EtOH on sample
- Vortex and incubate 30 min at -20°C
- Centrifuge 15 min at 20000 g
- Remove supernatant
- Wash with cold EtOH
- Vortex and centrifuge 15 min at 20000 g
- Remove supernatant and let dry at room temperature
- Resuspend in appropriate buffer
Methanol Precipitation of Triton X114 1%(w/v) solubilised samples
- Add 9 volumes of cold methanol.
- After a night at -70 °C, centrifuge at 12 000 g for 10 min.
- The pellet is resuspend in an appropriate buffer.