Hi guys, hope all is well,
so I just finished bradford assay on three different cell lysates. The chosen protein concentrations after analyzing the results were 3.5362 ug/ul, 0.6579 ug/ul, and 2.8289 ug/ul. My question here is when preparing the samples for loading prior to gel electrophoresis, what criteria should I consider in order to determine protein concentration in wells, sample volume in each well, ... etc. Listing examples would be very greatly appreciated. Many thanks for taking the time to read my question.
I'm not sure I get your question, but normally one tries to load 10-30 ug of protein per well. Depending on the well size, only a certain volume can fit, typically in large wells 25-30 ul. In your samples one has very low concentration, if you fit 30 ul in the well, 24 will be the maximum of your extract you can load (6 ul will be the 5x loading buffer), which means around 15 ug of protein. That's what you have to take also for the other samples (which will mean using only around 4.5 and 5.5 ul), but to have a uniform stacking and run I would suggest you to dilute them all in the same final volume.
I'm not sure I get your question, but normally one tries to load 10-30 ug of protein per well. Depending on the well size, only a certain volume can fit, typically in large wells 25-30 ul. In your samples one has very low concentration, if you fit 30 ul in the well, 24 will be the maximum of your extract you can load (6 ul will be the 5x loading buffer), which means around 15 ug of protein. That's what you have to take also for the other samples (which will mean using only around 4.5 and 5.5 ul), but to have a uniform stacking and run I would suggest you to dilute them all in the same final volume.
I think Pietros answer really answers this question as best you can without knowing what specific proteins you are using and for what purpose
By way of example and to elaborate on Pietros excellent answe, for Western blotting, I tend to load about 10ug if I know nothing about expression levels of protein in lysates
For highly expressed proteins e.g. metabolic genes you can go below 10ug and for low levels of expression I would defnitely recommend more; Infact up to 50ug although as Pietro has said 10-30ug is the general optimal range
As also said to some extent what you load depends on conc. and well volume (~ 20ul for safe loading of say a 20 well gel). To maximise loading volume try loading from the bottom up using flattened duck bill tips rather than top down with standard tips
Finally, bear in mind that large (>30ug) amounts of proteion derived from highly concentrated cell lysates can be viscous in the absence of sonication or shearing through narrow bore needle ( to break up genomic DNA)
Remember to add mercaptethanol or DTT and boil for 2-5 min after protian quantitation and before loading PAGE gels
Usually we load between 15-25ug proteins on each well sample two has low
Hi..
Load equal concentration of protein (10 microliter) of all three sample.
We usually use 15-30 ug of protein per well, if you want to compare between samples is very important that you load the same ug of protein in each well.
good luck
You have to ensure same conc (ug/ul) in all samples as first step, Then estimate how much sample volume(in ul) needed to be loaded in each well based on amount of protein you need to be run in the gel (Usually 20-40 ug). after that mix it with loading dye (1:1 or 2:1). take in mind that more than 40 ug per well is not recommeneded especially when you are going to do western blotting after electrophoresis before not of your protein will be blootted successfully
Good Luck
Including the amount of your sample you can also dilute the sample with the sample buffer in 1:1 &1:2 dilution prior to load on SDS-PAGE.
The experts have provided you excellent guidance. In our lab (CJ Lowenstein) we always do serial 2-fold dilution analyses of all protein samples and select values that fall on a straight line. This insures that inhibitors of the protein assay do not obscure the true concentration. We also prefer the BCA method because of reduced interference of extraction reagents.
If it is not clear to you, each sample should contain the same volume/ratio of loading buffer.
Does anyone disagree with this assertion?
You must run similar concentration of each sample in the each well if you want to compare the existance and pattern of proteins bands.
However if you want to compare the expression and amount of the protein in each sample, it is neccessary to load similar volume and after electrophoresis you can compare the bands of proteins by densitometry.
We commonly use 1ug/ul in our laboratory and find good results.
Our loading volume is usually 20-25 ul.
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