18 replies to this topic

[Prep!]

hey people....
i m facing this trouble of artifacts only in reduced samples!! is it a common problem? how to eliminate it?
i m using all high quality chemicals (electrophoresis grade!!) from sigma...
i tried DTT as well as BME and both are gicing me such artifacts... its difficult to analyze gels in this fashion!!
please suggest!!

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[mdfenko]

the bands are most likely an artifact that shows up more and more as the  reducing agent ages. it has been identified as, most likely, keratins  from dust.

[Prep!]

mdfenko, on 10 September 2010 - 07:44 AM, said:

the bands are most likely an artifact that shows up more and more as the  reducing agent ages. it has been identified as, most likely, keratins  from dust.

yeah i m pretty sure its an artifact but the catch is it has been prepared fresh!!!

[mdfenko]

is the 2-me (or dtt) fresh?

dust in the apparatus or glassware or dipping your finger in the buffer can also cause the artifact (only in the samples with reducing agent).

[Prep!]

yeah md.. its DTT and its fresh.. i tried with 2ME also and its still giving those artifacts... we also tried heating at different temperatures (60-100) and still the artifacts are not going!!! now i m going to try reducing without heatig.. say at 37 degrees for a longer incubation period.. atleast tat way i can confirm if heating is causing those artifacts...
also in the mean time i will try and get a fresh bottle of DTT to see if the reagent is causig any trouble but tat might not be possible immediately!!!

Edited by Prep!, 17 September 2010 - 08:34 PM.

[HomeBrew]

Run a mock control -- have everything you usually have your reduced samples, except add a volume of water equal to the volume of protein you usually add.  Run this no protein added control, and see if the artifacts appear.  This will tell you whether the artifacts are a consequence of reducing your sample, or whether they originate in your reagents.

[Prep!]

yeah tats a good one... why dint i think of it... thanx hb... will get back with the results!!!

[Prep!]

i did the control run without the protein samples and i did get artifacts in tat too.. so now i m sure that the problem lies with my DTT. I now filtered the freshly prepared DTT stock and used and still got artifacts in the gel!!! I also tried useing two different lots of DTT and still the same result!!!
Do i have to just wait for the fresh DTT reagent??!!! is the expiry period of DTT in powder form just around one year at 5 degrees??!!!

[mdfenko]

your source of the artifact may be dust. you can try cleaning the apparatus and prepare fresh solutions. make sure you don't dip your finger into any solution, that can cause or exacerbate the situation.

[Prep!]

hi md... the thing is the only difference between the reduced and non-reduced sample is the addition of DTT in the sample buffer... apart from that everything is same right from the stock solutions of gels till the running buffer and even the staining solutions!!!! that leaves my scratching my head!!!!

[HomeBrew]

Since the basis of silver staining is that the silver ions in solution are reduced to silver metal in the presence of proteins, I wonder if the reducing agent in your samples is causing metallic sliver deposits to appear as background.  If this is the case, it's not due to the purity of your agents, but perhaps due to the quantity used?

[mdfenko]

Prep!, on 21 September 2010 - 07:28 PM, said:

hi md... the thing is the only difference between the reduced and non-reduced sample is the addition of DTT in the sample buffer... apart from that everything is same right from the stock solutions of gels till the running buffer and even the staining solutions!!!! that leaves my scratching my head!!!!

that's right, the artifact is released by the reducing agent. we eliminate the artifact by omitting the reducing agent from our samples (where we can) or by using freshly purchased reducing agent to prepare the sample.

i can't locate the paper but we found a procedure that uses sodium metabisulfite to eliminate the artifact (it didn't appear to work in our hands, we may have applied it incorrectly).

ultimately, we explained the artifact (with references) and ignored it in our publications.

Edited by mdfenko, 22 September 2010 - 06:37 AM.

[HomeBrew]

mdfenko, on 22 September 2010 - 06:36 AM, said:

i can't locate the paper but we found a procedure that uses sodium metabisulfite to eliminate the artifact (it didn't appear to work in our hands, we may have applied it incorrectly).

Maybe it was this one (see at the bottom, just above "Anticipated Results")?

[Prep!]

hey guys what do ya suggest... what shud be the final quantity of DTT in the sample... right now its 50 mM in my case... and the loading amount is 5 micrograms. I will decrease the concentration of DTT and see its effect... I dont wanna lose the imputiries too if they are present!!!

[mdfenko]

HomeBrew, on 22 September 2010 - 07:37 AM, said:

Maybe it was this one (see at the bottom, just above "Anticipated Results")?

not that one, it's too new and is showing a destaining solution for silver stained gels (in the section that you cite), not something that will prevent the artifact.

if i get the opportunity i'll search for the paper(s) and give the reference (i have xerox copies, not electronic).