9 replies to this topic

[ulujm]

Hi
here a pict of my sdsPage after coomassie.

you can see the weird shape of the sample after migration.

so what is it?
the sample were sonicated many time (so no DNA).
protein loaded was 50ug.

any help

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[mdfenko]

in what buffer is your sample (prior to addition of laemmli sample buffer)?

salts and detergents and other additives can cause this.

[ulujm]

in RIPA classic buffer  even in commercial buffer I got this

[mdfenko]

the detergents and salts in the buffers are the most likely culprit.

you can try removing them or precipitating the protein away from them.

or

you can try loading a lot less. then you can silver stain to visualize your protein pattern.

[ulujm]

I doubt this is a buffer problem I try many differents buffer from my colleagues and this happen in 50% of the case.

[madrius1]

Can you give out the recipe of your RIPA buffer?

Because I would agree with mdfenko..

[ulujm]

TRIS 25mM
Triton 1%
Nadoc 1%
NaCl 150mM
EDTA 1mM

[madrius1]

Well, unless there has been an error in the making of the buffer, this is ok.

Is you protein ladder doing the same weird things?

[ulujm]

no the ladder is ok

so it's a sample problem but I don't know what it is

[mdfenko]

i've seen these effects in gels when too much salt or detergent (or other surfactant) is present in the sample.

your ripa may be okay but your sample load volume may be contributing too much salt and/or detergent to the well.

try reducing your sample volume.