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SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


gel loading question

gel loading question - SDS-PAGE Gel Electrophoresis Forum

gel loading question - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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  #1  
Old 08-08-2009, 04:26 AM
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Default gel loading question



I am running the gels for the Western Blot.
Usually, I combine the volume of my sample that has 20 ug of protein with double volume of buffer, then heat the samples, then centrifuge them and load into wells.

I find it hard to get every little drop out of the tube when I load samples, and I was curious if I can do this:

Instead of mixing, for example 4 ul of sample and 8 ul of buffer, like I would usually do, double my volumes and mix 8 ul of sample and 16 ul of buffer, vortex them, heat, centrifuge, ect,
But when loading pipette 12 ul (e.g the original volume that I was supposed to use).
Thus I donít have to worry that I am leaving stuff on the walls of the tube, and I can rinse the tip of the pipette as well to get the accurate amount.

Will that trick be ok? How do you guys solved this issue for yourself?

Thanks!
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Old 08-12-2009, 12:50 AM
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Default Re: gel loading question

I suppose what you suggested should be okay, since the ratio of the sample to buffer will still be the same and the concentration will not alter. There is always a slight problem with getting everything out of the tube, but I don't suppose a lot of people bother with that (at least I don't) unless the amount left in the tube is very significant. I have realized that some pipette tips are better for gel loading, and it may be due to the material it's made of.
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Old 08-12-2009, 02:48 AM
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Default Re: gel loading question

Thanks Jiajia, I actually tried to do it in the way that I have described earlier, we'll see what happends.

Apparently, I was told that it is a good practice not only because of the reduced mistake in pipetting, but also because samples evaporate less when heated.
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Old 08-13-2009, 01:14 AM
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Default Re: gel loading question

cool. do let us know how things turn out for u. =)
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Old 08-15-2009, 04:11 AM
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Default Re: gel loading question

It did not worked out too good, however I have no idea weather problem was with this method of sample prep or other things, such as gel casting or running the gel.

Heh. Will keep working on it.
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Old 08-17-2009, 03:07 AM
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Default Re: gel loading question

if it did not work out too good, i don't suppose it was caused by the amount of samples you put together with the loading dye since the proportion is the same. check out other aspects and let us know if you need some suggestions.
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  #7  
Old 08-17-2009, 12:00 PM
tgd tgd is offline
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Default Re: gel loading question

Hello,

Your calculation is absolutely correct, and should work, but perhaps is a little wasteful of precious sample?


1. If your original preparation is 20 ug/4ul (20 micrograms per 4 microlitres), this it the same as saying it it 5 mg/ml (5 milligrams of protein per milliliter of solution).

In your original protocol you diluted this 1 in 3 (1 plus 2) with sample buffer, that is 4ul (or 20 ug) of preparation was diluted 1 in 3 with sample buffer, giving a new solution with a protein concentration of 20 ug/ 12ul (or 1.6666' mg / ml). Obviously, if you load 12 ul, you are loading 20 ug of protein. The difficulty, of course, is getting a full 12 ul into the gel well.

2. If you take 8 ul (equivalent to 40ug) of preparation, and add 16 ul of sample buffer, you create a solution with a protein concentration of 40 ug/24 ul, *** AGAIN 1.6666' mg/ml ***. If you take 12 ul of this and load it on the gel, it is ONCE AGAIN equivalent to 20 ug. You can now load this quite cleanly, but you have wasted (perhaps) up to 20 ug of precious sample.

3. How about this? Lets say that you want the final volume of the diluted solution to be 20 ul, or thereabouts, and that you will load 12 ul of this (cleanly) and are willing to disgard what is left.

Then

Your preparation is 5 mg/ ml

You want to diute 1 in 3 with sample buffer, where the final volume is 20 ul.

So...

1ul in 3 ul is a 1-in-3 dilution;

and therefore

6.66 ul in 20 ul is a 1-in-3 dilution

But lets work in round figures:

7 ul in 21 ul is also a 1-in-3 dilution

4. Therefore, take 7 ul of your preparation, add 14 ul sample buffer. This solution is 1.6666' mg/ml. Load 12 ul of this, which will contain 20 ug.

You are losing 8 ul, or thereabouts. (You may wish to cut down further!)

5. Finally, two caveats. I have assumed that volumes are additive (if I add 8 ul of sample to 16 ul of buffer, say, the volume of the new solution is 24 ul). This is usually the case with dilute aqueous solutions, and we can assume that '1 plus 2' is the same as '1 in 3'. (This might not always be the case, especially if you are dealing with organic solvents!).

Secondly, I have ignored volume changes which may occur during boiling of samples. If, say, you boil for 3 min and the volume decreases appreciable, your 12 ul may contain significantly more protein.

Good luck, and I hope that this is clear. You raise a very good point.

Tgd
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  #8  
Old 08-18-2009, 11:55 AM
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Default Re: gel loading question

Dear Tgd, thank you for your time for answering!

Your explanatoins are very clear and make a lot of sense.

Today I will try this method again and see how it will work out. I am using the same sample, so there is no chance of different protein expression within it.

Will tell how it works :-)))

Thanks again!
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Old 03-21-2010, 11:02 AM
N27 N27 is offline
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Default Re: gel loading question

Hi,

I have 0.1ug/ul of protein and require 15ug to load on, which is calculating to 150ul to load on which is too big of a volume. What do i do?
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  #10  
Old 03-22-2010, 11:40 PM
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Default Re: gel loading question

Um, I am not sure what you can do with this sample now, but next time when you are going to perform lysis just use significantly smaller amount of lysis buffer, something that will give you 2-4 ug/ul for example
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