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Addition of sample loading buffer, before or after heating


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#1 2xzwei

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Posted 27 September 2012 - 05:10 AM

Hi!

I'm relatively new to Western blotting. During the preparation of my last blot I had a very strange event.

I added to a 20 µL concentration-normalized sample (in standard lysis buffer) 4 µL of 6x loading buffer (containing ß-Mercaptoethanol, SDS, Glycerol, Bromophenolblue in Tris-HCl pH 6.8) and heated it for 10 minutes at 95 °C. Afterwards I had a small blue pellet on the bottom of the tube and I wasn't able to resuspend it again (very viscous like gum). I just got few small insoluble pieces.

My colleague sticks to the same protocol and said she never observed this, but I should try to load the sample anyway. There was the problem that some of the pellet flakes were drifting out of the loading pocket. What has happened and how can I avoid this? Is it possible to heat my sample and then add the loading buffer afterwards or does it have to be added before heating?

Thanks for any recommendations...

#2 jmiller623

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Posted 27 September 2012 - 10:26 AM

Did you cool your samples afterwards? It sounds like SDS or DTT may have precipitated out. I usually heat up my loading buffer beforehand at 42C, vortex it, and add it to my samples to prevent this from happening. You should add the buffer beforehand and then heat your samples.

I hope this helps!

#3 mdfenko

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Posted 27 September 2012 - 11:51 AM

10 minutes at 95C may be causing denaturation of proteins in your sample. 5 minutes should be sufficient (or 10-20 minutes at 60-70C).

then again, the pellet may be due to carbohydrate.

if you get the pellet again, then load the supernate only.
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#4 2xzwei

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Posted 27 September 2012 - 10:59 PM

OK thanks for this information. I put the samples on ice after heating, just shortly for approx. 2 minutes before pipetting into the loading pocket. So I should not care about the pellet and all the protein is in the supernatant?

Another question:
I read that ß-ME should always be added shortly before using the loading buffer. Is this also true for DTT? Does it make any difference whether I use ß-ME or DTT?

#5 mdfenko

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Posted 28 September 2012 - 08:35 AM

depends on how you store the loading buffer. dtt and bme can both oxidize over time. so, it wouldn't hurt to treat the loading buffer the same way regardless of whether you use bme or dtt.

dtt is a stronger reducing agent than bme so you can use less for the same effect but, just follow recipes given for loading buffer with dtt. the buffer systems are mature.
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#6 jmiller623

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Posted 28 September 2012 - 08:54 AM

DTT just has a shorter half life but both are sufficient reducing agents. I use DTT because BME is volatile and after heating at 100 degrees, the smell kills me. You can add BME or DTT to your stock solutions and make sure that you keep the stock frozen until use. I usually keep about 500ul in the fridge and heat it up to 42 right before I add it to samples. I boil my samples at 95/100 for 5-10 minutes. Also to an elder, he had flakes and not goop, which wouldn't suggest protein/carbohydrate.

I think your problem is that you cooled your samples on ice which caused SDS to precipitate out. Don't cool your samples on ice afterwards if you are loading. You may want to let them cool closer to room temp but even this isn't necessary. Good luck and I hope this helps!




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