Hello,
Today I want to ask about sample buffer in western blot.
(For me, I use 4XSB as sample buffer)
if sample buffer melts in room temperature, is it okay to use it without
any treatments such as vortexing or tapping?
I am concerned that components in sample buffer remained in the bottom
so the concentration of such components
would be different between protein loading sample
if i do not vortex or tap melted sample buffer.
But in another view, i think that absence of mixing sample buffer itself
(by vortexing, tapping, etc)
seems to be not a critical problem because i do not detect any troubles
in my results of Western blot. I think that if it is really a critical problem,
I expect that the expression of loading control will not be the same level
in detection when considering the fact that I load same amounts from all of
my samples because same amount means same effect of components
of sample buffer.
I want to ask opinions from others who are more advanced than me is
Western blot.
Thank you!
I'm assuming your asking about your sample buffer that you use for loading your samples after lysis, is that correct? Here's what I do:
1. I bring my samples out of the -20 and allow them to sit on wet ice then thaw them a little with my fingers. 2. Once they are thawed, I vortex them briefly then hold them up to the light to make sure I don't see any darker areas in the tube that could signify ice or aggregates of protein. 3. I heat at 70C for 10min (this is for the NuPage system from Invitrogen so your temperatures and times may be different). 4. Quick spin to bring down condensation and load.
You may or may not notice any difference in your westerns if you don't vortex or mix, but I've seen myself that samples can have settled out chunks that go back into solution with some vortexing, which is why I always vortex before loading.
You can use samples that have thawed at RT, I've done it before; however, do be aware that protein degrades quickly even with the protease inhibitors that should be in your samples, if they aren't kept relatively cold at all times.
Dear Jenny Lynn Pokorny,
Thank you for your great answer with valuable information.
But i want to make sure something in my question above.
What I described as 'Sample buffer' is not the sample in which proteins are mixed with sample buffer. I am just mentioning sample buffer itself, which is blue-colored.
So my question is whether vortexing or tapping or other methods for mixing
sample buffer is necessary before mixing with proteins.
So far it seems to me that it is not critical issue, which means that i do not need to do such step but i want to ask related opinions from others.
(After mixing blue colored sample buffer with proteins,
i always vortex and spin down and i think it is very critical.)
Ok I see. I think how you store and use your sample buffer before your proteins are added to it is determined by the manufacturer's recommendations. For the NuPage buffer I typically use, I store it at RT and add it directly to the samples without mixing of any sort. I've never found this to be an issue when the buffer is at RT; however, if this particular buffer is cold at all it will be thick and chunky and need to be mixed thoroughly before use. I'd say as long as you're getting good results, keep doing what you're doing but you likely don't need to mix your buffer before using it. Of course, you can always hold it up to the light to make sure you aren't seeing any areas of thickness or heterogeneity in your buffer before you use it.
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