Hello everyone,

Currently a student in my first year of PhD, I am reaching out to everyone with experience on pull down assays with m7-GTP Agarose beads and/or mass spectrometry.

Right now I am working on a project that aims to characterize the composition of cap-binding complexes in Arabidopsis under different experimental conditions. To address the question of what proteins are susceptible to form cap-binding complexes, I am performing a cap analogue pull-down assay on protein extracts using m7GTP (C10-spacer)-Agarose beads (jena bioscience). The ultimate goal is to identify the eluted proteins by a LC-MS/MS analysis.

In order to focus only on proteins that bind the cap analogue specifically and leave out all proteins interacting the agarose matrix, I am also incubating the protein samples with unmodified agarose beads (jena bioscience). These « background noise » proteins will be removed from the MS analysis as non-specific interactors.

Following the pull-down assay, I did a silver nitrate staining of eluted proteins migrated in SDS-PAGE gel from both conditions (i.e proteins incubated with m7GTP-agarose beads and unmodified beads) to reveal their protein profile.

As you can see in the attached image, it seems that the protein profiles are very similar between the samples eluted from beads with or without cap analogue. I can not observe any clearly specific bands for samples eluted from m7GTP-agarose… I think my assay needs to be optimized to obtain a better ratio between « background noise » proteins and specific m7GTP-interacting proteins to facilitate the MS analysis and obtain biologically interesting results.

Has anyone encountered a similar situation ? Do you have any suggestions on how to minimize the proportion of non-specific proteins while preserving specific cap interactors ?

Here’s the protocol that I used for the experiment :

Total soluble proteins were extracted by grinding 100 mg of plant leaf tissue in 0.5 mL extraction buffer comprising 40 mM HEPES/KOH pH 7.6, 0.1 M KCl, 1 mM dithiothreitol, 1% phenylmethanesulfonyl fluoride, 10% glycerol and protease inhibitor cocktail (Roche). After centrifugation at 13000 rpm for 10 minutes, the supernatant was incubated with 100 uL of pre-washed Immobilized γ-Aminophenyl-m7GTP (C10-spacer) beads (Jena Bioscience) or unmodified agarose (Agarose blank, Jena Bioscience) at 4°C for 16 hours on a roller. The beads were subsequently centrifuged for 1 min at 13000 rpm and washed four times with extraction buffer at 4°C. The proteins linked to the cap analog or the unmodified agarose were eluted using 80 uL Laemmli 2X buffer and boiled for 5 min. Samples were conserved at -20°C.

Thank you in advance for your answers !

P.S. Here are the product IDs of the

m7GTP (C10-spacer)-Agarose beads:

https://www.jenabioscience.com/proteins/purification/nucleotide-binding-proteins/immobilized-guanosine-nucleotides/ac-155-immobilized-gamma-aminophenyl-m7gtp-c10-spacer

and the unmodified Agarose beads:

https://www.jenabioscience.com/proteins/purification/nucleotide-binding-proteins/unmodified-agarose/ac-001-agarose-blank

that I am using in my experiments.

Delyan