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  2. → Viewing Profile: Posts: laetitiaN

laetitiaN

Member Since 15 Jan 2013
Offline Last Active Feb 12 2013 04:12 AM
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Posts I've Made

In Topic: western blot troubleshooting

16 January 2013 - 08:27 AM

and the different sample come from different cells,
first picture on the left it's HUVEC and on the right its MDA (controlnegative  and different concentration of plant extract)
second picture HUVEC 2D culture - HUVEC 3D culture - MCF7 - MDAMB231 all are control negative

thanks

In Topic: western blot troubleshooting

16 January 2013 - 08:21 AM

hi,
thanks for your explications

my protocol is :
concentration gel 12% acrylamide
protean II ("big gels")
  • 13.6ml water
  • 10ml tris 1.5M pH 8.8
  • 200µl sds 20%
  • 16mL acrylamide (BIORAD 30% acrylamide and bis-acrylamide solution, 37.5:1)
  • 200µL APS 20%
stacking
  • 12.2mLwater
  • 4.6mL tris 0.5M pH 6.8
  • 100µl sds 20%
  • 2.66mL acrylamide
  • 300µl APS
  • 30µl TEMED
sample (dry cells pellets)  lysis in leamnli 2X
1 million of cells in 100µL,
boiling 5mn à 100°C than keep on ice
sample : 20µL (we process a bradford test and we have aroud 30µg of total prot in 20µL)
migration : 200V during 3H
transfert : semi dry 25V - 45mn
blocage : PBS, milk 5%, tween 0.1% 1H à Tamb
wash : PBS 0.1% tw, 5 times 5mn
hybridation : 2X 1h30 à ambiant T with 5 rince between
révélation: ECL than ECL+

I hope it helps to clarify with me those troubleshooting

thanks you again

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