I will be performing an IP using my desired antibody. What should be the concentration of the proteins (eluted from the beads) that will be used for LC-MS/MS ? The experiment will be done on HEK293T cells.
No one can answer this question as it depends entirely on the LC-MS METHOD used (conditions, column, settings etc). Using an LC-MS/MS system is not the same as using a basic lab instrument such as a spectrophotometer. Please contact an experienced LC-MS chromatographer at your institution (or outside lab) who can assist you in the preparation of your sample(s) and also the development of a selective method of analysis. Sample detection by MS is not "universal". Detection depends on many settings, parameters, the sample's characteristics and the METHOD used. Once the method has been established and a loading study run, then you will know what concentration and volume to use for your analysis. This process will allow you to obtain better quality data for analysis.
Hi Sharad,
You can easily purchase standard peptide mixtures generated from eg., HeLa cells. On a Thermo QE HF, coupled to nanoLC, I typically use 250ng of this mix to QA_QC our mass spectrometer. With DDA or DIA methods, you should be able to establish how many proteins you are identifying with such amounts of starting materials.
Once you have established your instrumentation optimization, start with your IP experiment optimization; you can have an estimate of the protein content of your HEK 293 cell lysate with protein assays. This could be accompanied by SDS-PAGE which is also visual and can help in estimation of losses. During IP loading, establish your flow through (proteins not binding to the column), washes, and finally, eluted from beads by using a fraction of the samples comparable to the starting volume of the lysate.
At the end of the day, if your immunoaffinity purifed complex is visible with colloidal Comassie Blue stain, you have enough materials for LC-MS/MS identification of the proteins. Be careful not to elute your proteins with eg., sample buffer. Harsh conditions that can elute protein complexes from an antibody column will introduce light and heavy chains of IgG into the final prep. The presence of these antibody chains will make your analysis difficult at the LC-MS/MS step since these IgG derived proteins are in vast excess in amounts compared to the affinity purified protein(s). If you are going to ID the final proteins by SDS_PAGE purification, you can avoid the light (~25kDa) and heavy (~50kDa) chains by excising bands around these proteins..
Good luck Sharad.
Hediye.
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