Hi everyone!
In a SDS-PAGE, what's the more effective way to separate high molecular weight proteinst (>100 kda) and get a good resoluion at the end? I know that the polyacrilamide gel concentracion can influence in the resolution as well as the voltage, but not exactly how.
Thanks! :)
You have right, you have to work with the concentration of the acrylamide in the gels or change the T/C ratio (total acrylamide to bisacrylamide). In first case you have to lower the concentration of acrylamide in the gel if you want to seperate proteins with HMW. How to find conditions? Look on the information from manufactures of protein markers - https://www.thermofisher.com/order/catalog/product/26619.
Of course lower concentration of gel, better resolution of high molecular weight proteins.
Voltage: if electrophoresis with boiling of proteins (standard) voltage generally do not influence the resolution, if you do not overheat the gels. Native conditions: electrophoresis run in cold room (fridge) or at least cold running buffer and tank surrounded by ice. Then room temperature is fine.
you try to check the gel / expired/ or concentration from stock bottle or something? I 40% acrylamine ?
I use to use 10% or 8% stacking gel and 5% resolving gel. If I need a good separation I would make 15 mm resolving gel and run it at ~80-90V. It already give your proteins start separation even you do not see it. When it reaches stacking gel I change voltage up to 100-110V. It don't generate to much hit and not soften gel. Also, It take longer time to run, but clearer separation. Usually I get very good separation and nice presentable gel.
pH is important to.
I agree with Violetta's answer.
I have separated on many occasions proteins of ~220-250KDa using 5% SDS-PAGE gels.
Be aware that:
- It is important to use a broad/high range pre-stained MW marker to enable you to decide when to stop running the gel.
- The 5% gel is quite fragile, so you need to be very delicate when handling it.
- Also for very large proteins, if you need to transfer by western to a membrane you may need to transfer for longer period of time for efficient transfer, and/or transfer to PVDF memebrane, rather than the nitrocellulose that is more frequently used.
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