i used the denatured collagen sample which was treated by 100 temperature, collagenase with 50U and trypsin with 0.1% for 10 mins, and put 7ul denatured collagen into each well. SDS-PAGE was running for at least 2 hours, with 220V and 0.01mA and 3w. However, i was confused why i got the bending bands? i guessed some of the reasons may be related the freshness of buffer, voltage and current, so could you provide me with some other clues?
Yes, excessive voltage could be the cause, try reducing the voltage to 120V.
Aissa Saidi THANK YOU for your suggestion ! i think i will conduct with the lower voltage.
The fact that your marker ran fine but the lanes are warped outwards tells me that your sample is way overloaded. You shouldn't load more than 20ug of protein into each lane, because otherwise your sample will have problems penetrating the gel. Also, it seems that the trypsin is not cutting the protein fully, resulting in this smear.
Just as a good measure, ensure that your tank is not leaking and you have filled the chamber with sufficient buffer to ensure an even current throughout the gel. The voltage you have chosen is a bit high, but running it at a lower voltage will not improve an overloaded gel.
Michael Adams Very appreciated with your detailed answers. I agree with you that the voltage was too high to run the protein smoothly. What's more, you also mentioned me that the efficiency of trypsin. i will try with the lower voltage this time. By the way, the loaded protein for each well was around 15mg. Here Just wanna confirm with you that the loaded protein unit should be "ng" instead of "mg"?
I apologise - it is neither! I corrected it in my original comment, but it should be no more than 20 ug (microgram) - this is the amount of protein that should be in your sample. So if you have 10mg/mL, then you should dilute your sample so that only the equivalent of 2 uL of your stock solution is loaded onto the gel. You need to measure your protein concentration, or at least estimate it. I recommend performing a BCA assay to determine the concentration, and only load a final amount of 20ug of protein onto each lane.
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