2 replies to this topic

[stefanie.grunwald@charite]

Dear all,

I am really frustrated because I do not ge rid of these strange bands. Can you help me please?
For protein isolation I used the following kit: Total RNA and Protein Isolation from cell cultures - NucleoSpin® RNA/Protein (Marcherey-Nagel 740933.50 - starting material: cell cultures from human skeletal muscle cells). The protocol I followed as well as the pictures I got after PonceauS staining are attached. The same bands/lanes appear in the gel (Coomassie) - I did not add these pictures. The only difference I performed using this kit was that I used DTT in the lysis buffer instead of 2b-Me.
I run the samples in TrisBis Gels 4-12% (see pictures) and TrisGlycine 8-16% (both from Invitrogen) - but it remained the same. Furthermore I applied the samples in 25µL volume for all of them and used fresh running buffers.Is it too less or too much salts or too less TCEP - the reducing agent? I am at a loss...

Thanks a lot!

Stefanie
 help-WB.pdf   259.86K   90 downloads

[scolix]

Sometimes if the sample is not homogenized properly, they clump and run in such a manner. Is that possible here?

[bob1]

I have seen this with native gels and I think the answer was too much salt, but it has been a couple of years since I was working with them.