Would anyone be able to let me know why my samples are running this way?
We ran it on 60V for 10mins up until the samples reached the gel and then increased the voltage to 100V. We used new running buffer, fresh protein lysates, 6x Lamelli buffer. Any solutions would be appreciated.
Dear Avanthika Priya Balu
Which is the % of your gel?
Which lysis buffer are you using?
considering that the protein marker seems to run well i think that is something present in your lysis buffer /eg an high % of guanidine? or some detergents? that may affect the running.
best regards
Manuele
My gel % is 4-15% gradient gel pre-made from BioRad.
I loaded 50ug protein on one side and 100uG protein on the other.
I use the RIPA lysis buffer from Sigma-Aldrich and contains 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0.
The question is, do your protein bands also look like this. As this is only the the migration front of the indicator it must not mean that your actual protein bands also run like this.
But if this is the case then the samples are not properly denatured. 50 -100 µg is a lot of protein so it is important to properly mix with Laemmli and denature everything at a sufficient temperature and time.
My bands are fine actually. They don't look like this. I do use 6x lamelli buffer and heat samples at 95C for 5mins before loading them.
Hi — “hill” bands usually mean the proteins never focused properly in the stacking gel. Common causes are overloading, high-salt/viscous samples (DNA/glycerol), poor stacking-gel polymerization, or incomplete denaturation. Quick fixes: reduce the protein load (try 1/3 the amount), boil with fresh DTT/β-ME and SDS, shear DNA (sonicate or Benzonase), dilute samples to lower salt/glycerol, and make sure your stacking gel and APS/TEMED are fresh. Run at low voltage for stacking (∼70–90 V) then increase gradually (∼120–150 V) for resolving. If the marker lanes look OK but your samples are still “hills,” the problem is in the sample (viscosity/overload/aggregation) rather than the gel or buffer.
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