I have a library of tagged proteins overexpressing cells and I have total cellular extracts for all of them but in small amount (around 500 ng of total proteins for each) and I would like to avoid defreezing the cells library to regrow them and reharvest largest protein samples. I would like to set up high throughput interaction assay of all these guys with a single protein. I thought about Gst-pulldowns, ELISA or Alpha screen technology. Also the protein samples are conserved in -20C, can the freezing alter their capacity to interact? I am looking for the fastest reliable technique.
Hi Pierre,
I consider it unfortunate you have not designed your assay the proper way right from the start, to create a matrix of clones suitable for e.g. split-ubiquitin (yeast) or split-YFP (yeast, plants, mammalian cells).
At the current stage, I think crosslinking would introduce only more artefactual interactions, not to mention possible protein degradation due to (re)freezing.
BUT, in case of some proteins (both soluble or hydrophobic), you may record some activation due to dilution, despite the (re)freezing inactivation.
Sorry I could not be of more help. Best of luck!
I agree with Marcin that it is unfortunate that you had not designed the assay before starting.
That being said, I think I understand from your description that you have tagged recombinant protein in total protein extract for each clone.
Since you want to ask about interaction with a single protein, you could set up a protein protein interaction by placing equal amounts of each of your protein extracts in a 96 well plate, then add your protein of interest to each well, dilute the reactions and shake at room temperature (if that is the expected optimal binding temp; if you are dealing with a mammalian protein, you might want to perform the binding reaction at 37C). After allowing binding, you will want to add the antibody to the tagged (if you have the antibody attached to a magnetic bead this would be terrific since it will make the following steps easier). If not, transfer all the samples to a fresh plate that contains the antibody to the tagged protein attached to the plate but free to interact with the incoming tagged protein. Centrifuge and remove all the supernatant, wash the plates at least twice, under salt conditions that favor maintenance of protein protein interactions and the antibody protein interaction. After the last wash, release the tagged protein and the interacting proteins with elution buffer. Examine by SDS-PAGE. All the samples should have the Tagged protein and only extracts from the clone that produces a tagged protein that interacts with your protein of interest should also contain the protein of interest. I presume that you have antibody for the specific protein you added to each well. A western with immunodetection with the antibody against that protein would be valuable in case the levels are too low for visualization by commasie.
If you can afford to use a magnetic bead system, that would make your life much easier. Check out the following reference and consider modifying as needed.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476752/
All the best
Hi Pierre,
I agree with Marcin, split-YFP/Ubi will make this work much easier.
At your current stage, maybe you can try just mix your tagged proteins together, and then do pull-down assay with your single protein and this mixture. If any protein can be pulled down, you can figure it out using MS.
Good luck.
You could give it a try with MST (MircoScale Thermophoresis). To do so, you would need to label your interacting protein. Then you fill each of your cell extracts into a reaction tube (or in a plate) and add the labeled protein. The reaction setups are filled into glass capillaries, and the measurement can be started.
During the measurement, a temperature gradient is induced which makes the molecules move (Thermophoresis). Their movement depends on different parameters that typically change when your molecule of interest (in this case your labeled protein) binds to an interaction partner (in your case this would be the overexpressed protein in the cell extract). You also need to perform negative controls with control cell extract (no protein overexpression) and in the best case scenario you also have a positive control.
In case you are not only interested if the two proteins are binding to each other, but you would also like to determine the affinity, you need to quantify the overexpressed protein. Then you need to prepare dilution series of the cell extracts. The change in thermophoresis is plotted against the ligand concentration; fitting the data points you will obtain the binding affinity.
Sure, freezing can change protein properties, depending on the freezing conditions (volume, speed of freezing, storage temperature, addition of glycerol etc.). We have best experience flash-freezing small aliquots (10µl) of pure protein in liquid nitrogen. For cell extracts, I guess this could be a different story.
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