Hi everyone. I am currently having problems in visualizing Ultra Low Range Molecular Weight Marker (M.W. 1,060–26,600) from SIGMA in Tricine-SDS-PAGE. I am following the protocol from Haider et al. (2018) and the staining method from SIGMA (Washing with water, fixing with 5% glutaraldehyde for 1 h, washing with water, staining with Coomassie G 0.025 in 10% acetic acid for 1 h and destaining in 10% acetic acid for 1 h).

I am running a 10% Tris tricine gel and loading 10uL/well of the weight marker diluted 1:20 in 1x sample buffer. I heat the diluted marker at 65 ºC for 2 minutes before loading. I use 90V continuos and minigels.

I am attaching a photo of the gel. The well in pink is the marker (I just see 3 bands) and the wells in yellow are my protein of interest, I am expecting 7 kDa. ¿Any suggestions to visualize the marker or to improve the technique?

Thanks in advance for your help and experience.

Notes:

-The 2x Sample Buffer is prepared as a solution of 100 mM Tris-HCl, pH 6.8, 1% SDS, 4% 2-mercaptoethanol, 0.02% Coomassie G, and 24% glycerol.