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.