Can anyone help me to explain while running my protein samples in SDS PAGE, Actin crosslinking doesn't make any significant increase in molecular mass comparing actin binding protein samples.
Hi Jijumon,
SDS-page breaks all (well , most) protein interaction. So, you don't see actin bundles or even filaments on a gel per se. The way to monitor bundling is to spi actin at ~17,000g for 15-20 minutes and carefully separate supernatant from pellet immediately following the spin. Bundled actin will go to the pellet fraction, monomeric and filamentous (unbundled) actin will stay in supernatant. See
Calcium binding is essential for plastin 3 function in Smn-deficient motoneurons.
Lyon AN, Pineda RH, Hao le T, Kudryashova E, Kudryashov DS, Beattie CE.
Hum Mol Genet. 2014 Apr 15;23(8):1990-2004. doi: 10.1093/hmg/ddt595. Epub 2013 Nov 23.
for details.
Cross-link briefly using 1mM DSP or a 4%PFA solution. Smaller complexes will run on SDS or BNP. Large complexes cannot be resolved by electrophoresis.
Instead of running on SDS-Gel try doing an In-solution digestion after which you can do a Mass spec to identify its binding or there is another method can perform an Intact protein identification using MALDI-TOF ( again intact protein and binding will depend on your mass, how many KDa is your protein?)
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