Hi，guys！
Does anyone know the difference between 2X and 5X sds loading buffer? It is said that maybe you cannot get appropriate results using 5X Loading buffer. Does anyone know the reasons?
Thanks a lot
zhiyuan
It is just the concentration of everything. I used 2x, 5x and 6x loading buffer myself and if I remember correctly, I prepared 10x buffer myself and I haven't had any problem with any of the loading buffer. Probably they didn't mix everything well with the 5x buffer because you don't have to aspirate it as much if you were using 2x loading buffer?
The 'X' represent here the multiple(times) concentration of any solution,
FOR eg. 1X is some value of concentration while 2X will be double the concentration of 1X and the 5X will be 5 times concentrated than 1 X.
In case of SDS loading dye we can use 2x or 5x just for tracking our sample how much they run, so the amount depends on the size of the well and the amount of sample which have to run over SDS PAGE.
the result do not very with loading dye concentration because it merely track the running path and indicates when to stop the power supply.
the final result can be concluded after staining and destaining procedure.
"In case of SDS loading dye we can use 2x or 5x just for tracking our sample how much they run" Are you sure? You are boiling your sample with this dye-SDS mixture to denature proteins in the sample. So, it is not just for tracking but also for denaturation.
Maybe we focus on two sides：one for tracking，and the other for denaturation
ya, in SDS loading dye, the beta merkeptoethanol which breaks the disulfide bond and denatures the protein, besides this heat plays important role in denaturation.
You will generally dilute stock solutions to 1x. As long as you dilute the solution appropriately (and the stock solution was made correctly), it shouldn't matter what the starting concentration was. I've used 2x, 5x, 6x, 10x, and 50x buffers, and they work the same because you dilute them to 1x.
I think one confusion that all of you have been having is that some of you are talking about the loading dye, and some are talking about the SDS running buffer. Whichever one you are talking about, just make sure the final concentration is 1x and you'll be fine.
Questioin is not clear.
is it about 'SDS-loading dye' or SDS-PAGE running buffer?
Hi, to some of you guys saying the "dye" could do reducing, denaturing or whatever. The dye is usually bromophenol blue which is not able to do anything to reduce your protein.
The Beta-mercaptoethanol or DTT which have active thiol groups which are the agent to "reduce" the disulphide of the protein (so the protein is not in their alpha or whatever form). The SDS is the thing "denature" the protein (so the protein is negatively and their migration "speed" is "proportional" to the mass of the protein during electrophoresis). It would be great if you guys look up what is in the buffer and understand what is electrophoresis and the function of each component in the system.
I used 20Ml from the sample added 5 Ml from (5x SDS loading dye) and the final concentration would be 1x
Ml = micro liter
Is your question about running buffer or sample buffer? If it's running buffer I always make 1x buffer and use it directly instead of dilution from higher strengths. Here is the recipe of 1x running buffer
1. Tris base - 18gms
2. Glycine-86.4gms
3. SDS- 6 grams
Make up to 6 litres with DD water.
1. I think we are discussing about loading buffer or sample buffer or loading dye not about running buffer.
I think question is, why we prepare or use different concentration of buffer (2X, 5X, 6X, 10X) when finally we use as 1X during final working solution?
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