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Service Protocoles

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.