western blot troubleshooting - (Jan/15/2013 )

hi everyone,

I'm a new PhD student in France, and it's the first time I post a question on a scientific forum.

I'm working on apoptosis in human cells MDAMB231, MCF7 and HUVEC. And I've got a big problem on my western blot results.
I add two exemples of results when I'm looking for actin.

I'v already try to denaturate again my samples in LAEMNLI solution 5mn at 100°C (test 080112) and it's a little bit better but not perfect.

Have you got any idea to explain those results ?

Thanks a lot

Laetitia

Hi Laetitia, welcome to the forum.

We need to know more about what you are doing in order to help you. The more details about your protocol (and the things you've tried) the better we can understand and look for answers

Welcome.

as almost says, we need more detail and specification of your problem. But it seems that you just have different amount of actin as a internal control. Thats because your loading conc. are different when you perform SDS-PAGE.You can calculate the amount maybe with densitometric methods.

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

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

Check your recipe for 2x Laemmli and the "dry" cell pellet - it looks a bit like you have too much salt in some of those samples. Also try running your samples a bit slower, 100-150 V should work fine and reduce the smearing you are seeing above the band. You shouldn't need to be lysing in Laemmli buffer to see actin, it is largely soluble and should show up well with an ordinary lysis buffer such as RIPA.

I would use a 10% gel for actin, but 12% should be OK.

You can reduce your washing to 3 or 4 x 5 min.

Sometimes these sorts of issues just come down to experience, eventually it will start working and there won't be any one thing that you can pinpoint that made it work.