Based on GST pull down assay, my protein A and protein B has interaction. i mix A and B and put in 4℃ overnight, centrifuge at 20,000g for 20min, then load on gel filtration chromatography with 100mM NaCl, but result reveal that there is completely no binding. so i wonder whether 20kg centrifugation or high pressure in gel filtration column can disrupt my protein-protein interaction?
Insufficient information for a good answer, it depends upon the conditions that lead to interaction between A and B. Also you didn't explain what you do after the 20000 g centrifugation, do you take the supernatant and do another pull down, or do you hope to see the AB complex in the pellet?
Just speaking theoretically, if one of the two proteins is linked to GST and you express both proteins in a biologically relevant environment (in cells, in a compartment where they are supposed to interact), you extract soluble proteins and you find that your GST pull down assay is positive (with cappropriate controls (i.e. GST only expressed with the other protein), then it is still possible that there is no interaction when you express A and B separately, purify them, and then mix them and incubate them together "in 4℃ overnight". May be they don't interact after simply mixing, may be the buffer in which you incubate doesn't allow interactions, may be they need another protein to do so.....I cannot tell.
Usually, pull down assays are done with low speed centrifugation (no more than 500 g) to avoid sedimentation of large protein aggregates that are non-specific. You only want your affinity beads to sediment, even 1 minute at 100 g is enough for that, and unspecific aggregates stay in the supernatant and are washed away. On the other hand, centrifugation at 20,000 g should not disrupt a specific protein-protein interactions, but you didn't explain why you do this in the first place. At 20000g all unspecific aggregates will sediment too.
Hello,
normally a 20000 g spin is not particularly high speed, the classical high speed goes up to 100 kg, and it does not disrupt complexes that do not precipitate with that force.
I think your problem might be different, as mentioned already by Jürgen, just a bit quickly. He is right that you don't explain how the GST pull-down is performed, but typically at least one of the two proteins is coming from a cell or tissue extract. Which means that the interaction you detect on the GST column might not be direct, so when you put together the two purified proteins they simply cannot interact.
If you are sure (by other means or information) that the interaction is direct, 100 mM NaCl is not particularly harsh on protein-protein interactions, normally problems start arising at 250-300 mM, unless there is a specific incompatibility of Na+ or Cl- with the specific binding... but this you should already know from the GST pull-down assay, which you carry on in a certain buffer, which I imagine will contain some NaCl. In any case it would be wise to do your direct binding in the same buffer in which you do the GST pull-down, since you know that the binding in those conditions occurs.
Finally, the gel filtration itself: I guess you are using an FPLC apparatus, are you sure you have the right SEC column? Also, can you be sure that the complex AB is sufficiently bigger than the single proteins (for example if A is large and B is only 1/10th of A, the complex AB might not be efficiently separated by A... at least it might depend on the type of SEC chosen). How do you check the fractions after chromatography, by WB or just Coomassie? Do you check for both proteins in all fractions?
I hope you have a bit to think about to solve your problem! Good luck.
Hi Zixun, I agree with Jürgen and Pietro.
20K x g centrifugation is relatively mild for protein complexes and should not disrupt high affinity protein interactions.
My main concern is that you are looking at protein-protein interactions with a Kd ~10-6 - 10-7 M and the gel filtration step is diluting the proteins past their Kd, hence no complex.
When you do a 20,000 g centrifugation, do you take the pellet or the supernatant?
I agree with Steingrimur. Compared to a pull down, which is typically done with small volumes of concentrated proteins, a big sizing column would dilute your protein significantly, so that they may not bind to each other because the Kd is too weak.
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