Hello everybody,
I accidentally prepared my samples with 5x laemmli buffer (final concentration in the samples), instead of 1x laemmli buffer. I didn't run the western blot yet, do you think this can be a problem? especially that I am interested to see a small protein (32 kda). I can dilute the samples to 1x laemmli buffer but I am afraid I don't have concentrated samples.
In my opinion, preparing protein samples using 5X Laemmli Buffer will not affect SDS-PAGE or western blotting. Actually the stock concentration 2X, 4X or 6X of Laemmli buffer do not indicate dilution factors, but they are just stock concentrations permitting correct addition of reagents to samples of high and low protein concentration. So, depending on your protein concentration in the sample you have to select the stock concentration of Laemmli buffer. This can be explained by examining the purpose of each ingredient of the Laemmli buffer.
The Tris-HCl maintains the pH of the mixture and SDS provides uniform charge to the polypeptide chain. Generally 1.4 µg SDS per binds to 1.0 µg polypeptide, so in 2X sample buffer, the SDS content is 4.0% (4 µg/µl) which is sufficient enough for protein concentration equal to 2.5ug/ul, but will be limiting for concentration higher than that. Similarly, the addition of β-ME or DTT is to prevent intramolecular S-S bond formation, so its concentration depends on the nature of your protein sample. Generally, 10% (2X stock buffer) is sufficient for whole-cell protein isolate, but higher concentration will not affect PAGE or western blotting. Presence of Glycerol is to impart density to the whole mixture which facilitating gel loading and also prevent the flow of sample out from the well, so higher glycerol will also not affect any downstream processing. Finally Bromophenol blue is for visualization (which imparts some charge to protein which is negligible compare to SDS). Moreover, during western blotting, the gel is washed by transfer buffer which removes any extra SDS in the gel.
As long as they are all at the same concentration it should be fine
You might find the mobility during electrophoresis is altered and your blue dye front is very intense; but other than that it should be ok
The dye front will definitely run ahead of a 30kd protein so even with increased intensity it should not obscure detection of your protein
High concentration of SDS dye buffer would interfere with the mobility of your sample. If you have more sample, i suggest that you dilute it with your sample. Boil the entire sample and load the required volume in SDS-PAGE. Remaining boiled sample could be stored at -20 degree Centigrade freezer.
I ran the samples and developed it. There was no problem, I got nice bands.
Thank you
- Badges
- Science method