When running a western blot, I usually load less ladder (6uL) than any of my samples (20uL). Someone recently told me that I should add Laemmli buffer to my ladder so that it is loaded at an equal volume to the samples. He said that doing this would make the ladder more reliable and less likely to run at a different rate. Should I do this, or should I just load 20uL of ladder? Does it really make a difference if different volumes are loaded in parallel?
Thank you.
For the best resolution always run the same quantity of protein in the same volume of sample buffer; for example 20 ug per well in each well and you have to calculate it so that the 20 ug of protein is in say 25 uL of sample. So you load 25 uL of sample in each lane and each lane contains 20ug of protein (I am just using these numbers as an example). I always run 2ul and 10 ul of the standards on the outer most lanes.
Hi,
proteins will need different amounts of space in your gel matrix depending on how much protein you load on your gel. If you load very low and very high amounts of proteins next to each other, your gel and therefore the pattern of your separated proteins will be effected. More protein will need more space. The proteins will deform the lane if in the next lane much less protein is loaded. Proteins with a high concentration within your sample can occupy so much space that their running time will not correlate with their size any more. Therefore it is important to load a suiting amount of protein and to load more or less equal amounts of proteins in every lane. The sample buffer can not totally even out the effects when you load much less protein in your ladder lane. It can help to prevent the distortion of the lane next to your ladder a bit but not if you load MUCH more sample. And the proteins of a highly concentrated sample could run slower (much protein hat to be pressed through the matrix) than low concentrated ladder proteins.
So it is not only a question of equal volumes. More important is the protein amount that has to run/squeeze through your gel matrix.
I hope this may help,
Kristin
Hi David,
The ladder is used just to estimate molecular weights of the bands you get in your sample lanes. Thus, it seems logical to load as less amount of ladder as possible. In running a gel, it is the total protein content (microgram) of the samples that should be adjusted to an equal amount (microgram) in order to have a reliable estimation of the amount of your protein/band of interest. On the other hand, whether you are allowed to add loading buffer to your ladder or not, depends on the type of the ladder you are using. Some ladders are ready-to-use ones and are loaded in the gel without the need for boiling or adding loading buffer ( which contains SDS and might interfere with the normal protein/SDS ratio of your ladder and affect the structure of the proteins in your ladder) to. Note the type of the ladder you are using before adding loading buffer. These ladders also contain various dyes attached to different proteins of their content and boiling process may detach the dyes from proteins. In the case of simple ladders you are allowed both to boil them and add loading buffer. Anyway, taking all of these into account, in my opinion loading the same amount of ladder (in terms of µg protein content) with two different volumes would not make any difference in their running rates and the pattern of the final bands you get because the protein contents are the same in both cases.
Good luck!
For the best resolution always run the same quantity of protein in the same volume of sample buffer; for example 20 ug per well in each well and you have to calculate it so that the 20 ug of protein is in say 25 uL of sample. So you load 25 uL of sample in each lane and each lane contains 20ug of protein (I am just using these numbers as an example). I always run 2ul and 10 ul of the standards on the outer most lanes.
The sample protein stacking and separation by the Laemmli gel depends on the pH and ionic composition of the stacking gel, the running buffer, and the sample solution. Ideally all samples should be approximately equal in volume, protein concentration and sample buffer composition. The stacking and resolution is created not only by differences in gel composition and sieving, however also by differences in pH and ionic strength between the Tris Glycine in the stacking gel, separating gel and running buffers. How this all plays out depends also on sample composition and volume. Instrument and well configuration (horizontal versus vertical); and how the electric filed is applied; all can be important.
Hi David,
It dose not matter whether you are dilute your protein ladder or not because the purpose of ladder is just for justifying protein weight. you do not need to add equal amount of ladder and your protein sample.
I also use 6 ul of the ladder dye and has been working for years, with the ladder without adding loading dye to it.
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