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SDS-PAGE sample streaking issue - (Apr/04/2013 )

Hi there,
This is my first time posting on a forum like this. My supervisor hasn't really been much help here so now I'm asking you all.
Basically I'm running gels of some protein extracts. I ran similar gels and protein samples over 12 months ago and those worked out fine. According to my records I am doing everything the same (sample gels, solution recipes, extraction technique etc) and I am getting horrible verticle streaking of my protein sample through the gel. I will include an example below.

The two middle lanes are a protein ladder.
The left most and right most lanes are my protein extract with the sample loading being less in the left lane.

Any help you can offer regarding this would be greatly appreciated. If it would help to know more informaiton about procedures please just ask.

Thanks

-littledamn-

Is this BN PAGE or just ordinary SDS PAGE?

Can you tell us what you have done -running voltage/amperage, amount loaded, % gel, loading buffer and lysis buffer component concentrations...

-bob1-

Hi,
Thanks for the reply. It's just an ordinary SDS-PAGE using mostly a commercial kit which is Invotrogen Tricine Gels and Buffers. This unfortunately means the exact recipes are difficult (if not impossible) to find.
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Protein-Gel-Electrophoresis/Protein-Gels/specialized-protein-separation/Tricine-Gels.html#comergent_product_list_67029


Gels are Tricine 16%
Total amount loaded is around 20 uL - protein concentration has not been measured for these but I'm confident there's not an excess of protein. I've tried running the same gel with the same protein sample with less protein added and still get the streaking.
Loading buffer - http://products.invitrogen.com/ivgn/product/LC1676
Running buffer - http://products.invitrogen.com/ivgn/product/LC1675
Extracted rotein is first acetone precipitated before being resuspended in the loading buffer + 1 uL of reducing agent - so there is no real lysis buffer.
Gel is run at constant voltage of 120V for 60-90 mins

Please let me know if I am missing anything. I hope this helps.

-littledamn-

OK, that should all be OK. I presume that the loading buffer is not expired? How much salt is present in the sample and is your reducing agent fresh?

-bob1-

There should be minimal salt as a result of the precipitation. There will be some as I know these protein bind some ions, but again it shouldn't be a large amount.

The reducing agent now you mention it is a bit old. Might as well order some more. I think our DTT (which I could use as a replacement) is also quite old.

-littledamn-

littledamn on Mon Apr 8 02:11:13 2013 said:

I think our DTT (which I could use as a replacement) is also quite old.

not a problem if stored properly (we store solid at -20C, lasts "forever").

(stating the obvious) you seem to have incomplete solubilization of your sample in the loading buffer (read "aggregates"). this may be due to insufficient heat treatment of the sample in the loading buffer. it can also be due to insufficient dispersal of the pellet in the loading buffer. or a combination.

residual acetone may also be affecting the gel.

despite your protests to the contrary, you may be overloading the gel as well.

the narrowing of the lanes that you are seeing is usually caused by salt, organics, lipids...

-mdfenko-

Alrighty then, what would you suggest I can do to fix this up. There isn't much I can do with the actual sample as for various reasons I can't mess with it. But would you suggest adding anything to this list of improvements?

-Leave open for longer after acetone extraction to make sure any residual acetone evaporates
-Increase amount of loading dye in comparison to sample. This increase SDS and hence dissolve any lipids that may be sticking around. And also perhaps dilute any other contaminants such as salt.
-Heat for longer
-Give it a decent spin after heating to carefully check for any aggregates. If there are some there, heat again. Spin again and only load the liquid (if there is still any pellet).

I've done some of the above but perhaps I could have done it more carefully.

Apologies about checking this. I only have a limited amount of sample and can't really get more so I can't really run a proper series of troubleshooting gels.

Again thanks for the advice so far.

-littledamn-

littledamn on Mon Apr 8 21:59:48 2013 said:


Alrighty then, what would you suggest I can do to fix this up. There isn't much I can do with the actual sample as for various reasons I can't mess with it. But would you suggest adding anything to this list of improvements?

-Leave open for longer after acetone extraction to make sure any residual acetone evaporates

yes

-Increase amount of loading dye in comparison to sample. This increase SDS and hence dissolve any lipids that may be sticking around. And also perhaps dilute any other contaminants such as salt.

you should maintain a constant ratio of loading dye to sample (based on the loading dye's concentration). you can dilute the sample with water and add more loading dye to maintain the ratio.

-Heat for longer

if you heat at 90-100C then you may already be heating for too long. heating too long at boiling (or near boiling) temperatures can cause protein aggregation, even in the presence of sds and reducing agent. you can, however, heat at 60-70C for 10-20 (or more) minutes to more completely denature your sample without aggregation.

-Give it a decent spin after heating to carefully check for any aggregates. If there are some there, heat again. Spin again and only load the liquid (if there is still any pellet).

yes, you should clarify the sample prior to loading to ensure that particulates are not introduced to the gel. you can also homogenize the sample in loading dye with a "pellet pestle" before heating. then clarify prior to loading (anything that hasn't dissolved by then probably won't).

I've done some of the above but perhaps I could have done it more carefully.

should i comment on this?

-mdfenko-

mdfenko on Tue Apr 9 14:44:49 2013 said:


should i comment on this?


If you like :D

-littledamn-