Hi, please have a look to the pdf. and check the concentrations of your buffer. maybe you have to dilute your sample more.

https://tools.thermofisher.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf

There are some Substances as MOPS which are not compatible with this kind of assay = n/a Denotes that the compound was not tested in this assay

Good luck

P.S. Try 660nm Assay

There is a carbocyanine dye known as "Stains All" that stains phosphoproteins blue and non-phosphorylated proteins red, if it is still available. If you run a colorimetric assay against a standard dilution of phosphoproteins with this dye, you should be able to determine the phosphoproteins in your samples by choosing a suitable red wavelength.

Alternatively, you could try using one of the commercially available fluorescent total protein assays. The SYPRO dyes, for example, don't suffer the same level of variation as the Lowry type assay's with different protein species, as the dye binds to a detergent shell (SDS) on the protein surface.

I get this problem with most primary human samples, in my case it is not due to our lysing buffer as our system works perfectly with cell lines.

Which loading control do you use?

Thank you for your responses, Natasha, that table is very useful, you are correct, 660nm looks like it is an option.

Andrew, those are both useful, I will look into them.

Victoria, I use a PanAKT mAb or beta actin as my loading control.

I have no experience with PanAkt but beta Actin is at best unreliable with our patient samples (even in samples from the same patient at different timepoints!).

I had better results with alpha Tubulin and always go for a ponceau staining of the membrane as I have found that this shows the proteins the best.

Thank you Victoria, good to know!