4 replies to this topic

[kylvalda]

Hi there,

got the following prob:

Run 9 samples (all processed the same way) in SDS Page under following conditions:

Nupage Bis-Tris 4-12% precast gels and MOPS buffer from Invitrogen
100-120V

The samples are cells washed once in PBS, lysed in RIPA + PIs, added Laemmli (reducing)
cooked 5min 99deg, chilled on ice and loaded onto gel

And what I get is 7 of 9 samples running very strangely on the gel (have a look at attached pic).

First thought samples were degraded but....

Only thing I didn't do is spin after cooking the samples

Or too high/low salt concentration?

HELP!

Attached File(s)

•  chrfexpr001.jpg (172.07K)
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[ElHo]

Hi,
looks like too much salt in the samles to me.

[mdfenko]

two possible problems, too much salt and crystallized sds. sds crystallizes at low temperatures (on ice). you should warm your samples back to room temperature and remix them before applying to the gel.

you can reduce salt in a small aliquot for sds-page by drop dialysis.

Edited by mdfenko, 17 September 2010 - 08:37 AM.

[GNANA]

to me it doesnt look lik the prob in the sample or its prep, but the prob is in gel casting or running condn.....

[bob1]

I have seen similar things with a colleague's gels using the same system. I think he reduced the salt in his preps and they improved.

GNANA, on 17 September 2010 - 02:43 PM, said:

to me it doesnt look lik the prob in the sample or its prep, but the prob is in gel casting or running condn.....

The OP is using pre-cast gels and a pre-made buffer system from Invitrogen, it shouldn't be either of the problems you mention.