Hi,
I recently got in an argument with a new coworker on how to dilute protein samples for WB.
I normally dilute the Laemmli based on the total volume that I want to load in the wells, Example: if I have 5uL of protein sample I will add 2.5uL of LAE( 1/4 of 10) and add 2.5 of H2O, and the 10uL resulted are loaded in the well.
He assert to dilute the Laemmli based on the sample volume.
Example; for 5ul of protein sample He will add 1.67uL of LAE (1/3 of protein sample) and then or load 6.67 in the well or take to a certain volume with H2O.
His argument is that on the LAEMMLI protocol is written "dilute 1:4 with sample" but I dont catch the reasoning for it to be like this, because like this, more concentrated protein sample will have less LAEMMLI than a less concentrated one but the ug of protein is the same.
Hello Domiziano Tosi,
Both of you are right because the main purpose is served namely, the sample loading buffer is finally diluted from 4X to 1X.
I fail to understand your reasoning 'that more concentrated protein sample will have less LAEMMLI than a less concentrated one'. Since both, you and your coworker have used 5ul of the same protein sample, the microgram of protein will remain the same. The purpose is to dilute the sample loading buffer to 1X. For example, if you were to use 2X Laemmli sample loading buffer, you would have to combine equal volumes of the protein sample and the sample loading buffer (say 5ul of protein sample + 5ul of 2X Laemmli sample loading buffer to give 10ul volume to be loaded on the gel).
Only in case if your protein sample is dilute, you will need a higher concentration of sample loading buffer so that you may use a higher volume of protein sample to dilute the sample loading buffer. For instance, you may have to use a 6X sample loading buffer instead of 4X.
Best.
Usually we focus on the amount of protein loaded into the gel rather than the volume of the load, the volume helps us to adjust the amount of protein sample. Therefore, when you want to load a protein, it is not necessary to add water if the sample volume is not very small. Be aware that the loading buffer depends on the volume, not the sample amount. If it depended on the amount of the sample, the protocol would have been that for every X ug of the protein x ul of the loading buffer should be added. So if you use 5 ul of protein and the volume of loading on the gel is either 10 ul or 15 ul, the result will be the same because the amount of protein remained the same. So you will have used more loading buffer unnecessarily.
Sure between both arguments, I think yours is the correct one, because he would add water after mixing the sample with the buffer
Malcolm Nobre I will give you an example of my experiments.
- I had 1 sample that to take 12ug I had to take 4.5 of sample, then with my colleague method I had to add 1.5ul of LAE (total volume 6ul). Another sample I had to take 1.5uL of sample and add 0.5 of LAE and add 4ul of H20(total volume of 6uL to load sample volume). So 1° sample LAE dilute 1:4, meanwhile the 2° sample the LAE was dilute 1:12.
- Instead with my method I would have done 1° sample 4.5+1.5LAE(6 total) and 2° sample 1.5+1.5LAE+3H2O (6 total). Both LAE dilute 1:4.
The reasoning "that more concentrated protein sample will have less LAEMMLI than a less concentrated one" is that in the first scenario both sample had 12ug of protein in the solution but one had a third of LAE, so in my head I thought that the second sample will be less denatured or will have more problem in the loading phase(less glycerol).
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