Hi Jessica,

You are right about the overloaded lanes. In your explanation you said you add b-mercaptoethanol but no SDS. I would suggest to use Laemmli buffer, as it contains SDS. SDS will charge negatively your proteins so the migration will be only dependent on the length of your proteins. Always heat your samples after mixing the Laemmli buffer with your protein sample.

Also, increase the amount of your protein standards (lanes 9-10) and protein markers (just in case). Probably you cannot see the lowest 1060 marker because it has not been resolved correctly, I guess the band you think is 3496Da is actually 3496+1060Da bands.

Xi Jiang Thank you so much for your response

I used the following sample buffer which contains 2% SDS

https://www.bio-rad.com/en-uk/sku/1610739-tricine-sample-buffer-for-protein-gels-30-ml?ID=1610739

Do you recommend to use Laemelli instead?

I will increase the amount of protein marker and run for longer too, thank you so much for your advice.

I did however, load 1mg/ml of protein standard so I am very unsure as to why it is not visible

Thank you in advanced

I just checked the composition of the sample buffer and in our lab we use a slightly different buffer, for 2x Laemmli we have 4% SDS (2% final when mixed with protein sample), 10% b-me (5% final) and we have less glycerol 20% (10% final). The rest of ingredients of the sample buffer are quite similar and not so relevant (I think) for the electrophoresis.

If you think your protein is not well denatured, consider using also urea or other chaotropic agent. Be careful with this, as 6M urea in the sample buffer will widen a lot the protein lane, to prevent this, add same amount of urea to all samples, even the marker.