ANTI-FLAG® M2 Affinity Gel from Sigma-Aldrich

March 11, 2013

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City of Hope
Molecular and Cellular Biology
PhD Student

Different concentrations of exogenous small RNA were transfected into cells expressing the FLAG-tagged RNA binding protein of interest. Top: Immunoblot verifying successful (IP lanes) and complete (supernatant) immunoprecipitation of our FLAG-tagged protein of interest. GAPDH serves as a loading control and verifies no non-specific immunoprecipitation of non-FLAG protein. Bottom: Northern blot, probing single strands of exogenously transfected small RNA. We see that the limit of detection is ~10 nM of transfected small RNA. The U6 RNA, which does not bind to the RNA binding protein of interest, serves as a loading control, as well as a specificity control.

Company:

Sigma-Aldrich

Product Name:

ANTI-FLAG® M2 Affinity Gel

Catalog Number:

A2220
Image

I needed to understand how exogenous small RNA duplexes load into RNA binding proteins (RBPs). We had a system with cells stably overexpressing FLAG-tagged RBPs. By transfecting the small RNA duplexes and immunoprecipitating the FLAG-tagged RBPs, we could then perform a Northern blot to monitor small RNA loading. The Sigma ANTI-FLAG® M2 Affinity Gel was an obvious choice. Our protocol calls for saving a small amount of sample at intermittent steps to confirm the technique is working both at the protein and RNA level. For simplicity, we employed a medium-stringent lysis and washing solution for all IP steps. Liberating the small RNA from RBPs was achieved by boiling in RNA loading buffer.

Experimental Design and Results Summary

Application

Immunoprecipitation (IP)

Starting Material

Cell lysates

Protocol Overview

Transfections were performed in 10 cm dishes. At day 0, cells were transfected with small RNAs. At day 1, cells were lysed in a small volume and protein concentration was determined. In my pilot studies, I did not have high background or non-specific binding during IPs, so I did not pre-clear the beads. Using P-200 tips that I had cut with scissors to slightly increase the tip mouth diameter, I simply centrifuged the necessary volume, removed the glycerol supernatant (~50% of the volume), and re-suspended in an equal volume of lysis/IP buffer. I used 20 ul of re-suspended beads solution (which is ~10 ul of beads) for a 2 mg input protein lysate sample in a total volume of 1 ml. Another ~40-50% of the unused lysate was added to RNASTAT60 (or TRIZOL) for input RNA for Northern blotting. After gentle overnight rotation at 4°C, samples were centrifuged, removed of supernatant, and washed with 500 ul lysis/IP buffer. The initial supernatant was kept as a control. After the final wash, my samples were centrifuged and supernatant removed. The pellet was ~20 ul. Using cut P-20 tips, I removed 3 ul of the pellet for immunoblotting (by boiling with SDS PAGE loading dye). To the remaining pellet, I added the appropriate amount of RNA loading dye for ~30 ul total volume, followed by boiling for 10 min.

Tips

See ‘Results Summary’ and ‘Additional Notes’ below

Results Summary

When immunoblotting, I used 30 ug of protein (~15% v/v) of input protein and all of the prepared IP’ed sample. Despite only being 3 ul of the pellet at the end of the IP procedure, I observed a very robust band using a rabbit (i.e., non-mouse) anti-FLAG antibody. I do not recommend using a mouse anti-FLAG antibody for immunoblotting, as the antibody may recognize the mouse antigens from the affinity gel and give a false-positive signal for your protein of interest. The supernatant did not show any signal, indicating complete IP. When performing a Northern blot on the IP’ed small RNAs, the dye on the IP lanes may appear to be migrating slightly slower than the dye from the input or control lanes, probably due to the fact that the IP samples still have some of the Sepharose beads from the affinity gel that retard the migration of the dye slightly. Visualizing the final gel, however, showed that all small RNA samples had migrated at the same rate. For the small RNA strand from the RNA duplex that is predicted to be bound and stabilized by the RBP, a clear band was shown. The strand of the RNA duplex that is not predicted to be bound by the RBP was faintly detected, if at all.

Additional Notes

If the stock bottle has become inverted and some of the affinity gel is stuck to the top of the cap, you can place the stock bottle inside a 50 ml conical tube and, using another water-filled 50 ml conical tube as a balance, perform a low speed centrifugation for 10 minutes to pellet all the affinity gel at the bottom of the stock bottle.

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Image Gallery

Different concentrations of exogenous small RNA were transfected into cells expressing the FLAG-tagged RNA binding protein of interest. Top: Immunoblot verifying successful (IP lanes) and complete (supernatant) immunoprecipitation of our FLAG-tagged protein of interest. GAPDH serves as a loading control and verifies no non-specific immunoprecipitation of non-FLAG protein. Bottom: Northern blot, probing single strands of exogenously transfected small RNA. We see that the limit of detection is ~10 nM of transfected small RNA. The U6 RNA, which does not bind to the RNA binding protein of interest, serves as a loading control, as well as a specificity control.

Summary

The Good

Simple to use. Can IP a large amount of FLAG-tagged protein of interest using only a small volume of the affinity gel.

The Bad

Stock solution is very viscous. May have to use cut tips when aspirating the affinity gel.

The Bottom Line

A very efficient product that was very straightforward for my experimental purpose. Highly recommended.

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