Strips and Arrays protocol

Echelon’s Lipid Strips and Arrays are simple, but highly effective and widely used assays for determining lipid-protein interactions. Here we provide an extensive protocol as a guide and reference for use with your protein of interest. The protocol includes suggestions for positive controls and buffers as well as general recommendations for a successful assay.

Protocol

This is an example protocol that has been optimized for use with EBI’s PI(4,5)P2 Grip (Cat # G-4501). Please modify as needed for your protein of interest. Additional control proteins are listed in the table below. We recommend reading the protocol notes before proceeding. 

1)Add controls (optional):
Spot 1 μL of PI(4,5)P2 Grip and 1 μL of secondary antibody directly onto open areas of the dry membrane. Allow the spots to dry completely before proceeding to step 2.This is a control for the HRP conjugate and detection reagent (see Assay Note 5).

2) Block:
Cover the membrane with 5 to 10 mL of blocking buffer PBS-T + 3% BSA and gently agitate for one hour at room temperature (RT) or overnight at 4°C.

3) Add Protein of interest:
Discard blocking buffer and add 0.5 μg/mL PI(4,5)P2 Grip™ protein in 5 mL PBS-T + 3% BSA, or enough to cover the membrane. Incubate for 1 hr at RT with gentle agitation.

4) Wash:
Discard the protein solution and wash with >5 mL PBS-T three times with gentle agitation for five to ten minutes each.

5) Anti-GST-HRP antibody:
Discard wash solution and add 75 μL anti-GST-HRP antibody (K-SEC2) to 5 mL PBS-T + 3% BSA blocking solution and incubate for 1 hr at RT with gentle agitation.

6) Wash:
As in step 3

7) Detect:
Discard wash solution and detect the bound protein by detection methods of choice e.g., Echelon’s K-TMBP, TMB Precipitating or similar, Chemiluminescent or ECL detection from KPL or similar. For K-TMBP, after discarding the final wash, add 1 to 2 mL TMB Precipitating (catalog K-TMBP) per membrane with gentle agitation.

If TMB is used, protein-lipid interaction will develop within 2-20 minutes with the spots turning blue. Stop the reaction by adding an ample amount of dH2O (≥ 15mL). Avoid overdevelopment to avoid high background. Development time for ECL should be similar. Record an image of the membrane.

Results may vary for a given protein depending on the blocking buffers used. See below for an example. If excessive or non-specific binding is observed or suspected, it is recommended to optimize the buffer conditions.

Blocking Buffer Optimization

Multi-PIP Grip (catalog G-9901) was used on PIP Strips (catalog P-6001) at 0.5 μg/mL in three different blocking buffers. Strips were developed with TMB Precipitating solution (catalog K-TMBP).

Buffers

These are suggested buffers for optimization. Recommended block solutions for control proteins can be found in the ‘Controls‘ section

Notes

1. During all incubation and wash steps, make sure the membrane stays wet and never dries. We recommend gentle agitation during all incubation and wash steps. We have found that protein-lipid binding can be affected by the blocking buffer, we recommend trying different buffers for each protein of interest, see table above for suggested buffers.
2. 1-2 μg/mL is recommended as a starting concentration for a new protein of interest. The end user must optimize protein concentration for each protein of interest. If high background is experienced or a protein interacts with multiple lipids instead of showing the expected specificity, we recommend decreasing the amount of protein used or running the protein in a different blocking buffer. We do not recommend incubating the protein overnight at 4°C. This may degrade the protein and cause a decrease in activity.
3. We do not recommend using cell lysate, only purified protein or antibodies.
4. The primary and secondary antibodies listed are used routinely at Echelon. Other similar antibodies are likely to work effectively in protein-lipid overlay assays.
5. We recommend including control spots. Test the secondary HRP antibody and detection reagent with the detector by spotting 1 μL of detector proteins and secondary detector on the strip before running the protein lipid overlay assay. The spots will show up strongly with the detectors, indicating that both are working.
6. Chemiluminescent or ECL detection may produce variable results depending on the kind of ECL used, exposure times, volume, film, and development analysis software. ECL is prone to loosing activity close to and after the expiration date labeled on the bottle. Avoid using ultrasensitive ECL reagents due to possible high background.
7. Please see EBI’s FAQ for Lipid Strip and Lipid Array Products for additional information and recommendations.
8. We do not recommend stripping and re-probing the membrane strips or arrays using Western/protein blot protocols. The stability of the individual lipid spots following such treatment has not been confirmed.
9. The blue blank spot contains traces of Xylene Cyanol FF which shows fluorescence around 615 nm and may be visible when using fluorescence based imgaing such as Licor.

Controls

FAQ

Echelon Biosciences’ Lipid Strips and Arrays are simple protein-lipid overlay assays used to screen for selectivity and relative affinity between a protein of interest and common cellular lipids. Our recommended protocol for these products can be found here. For additional troubleshooting, please see our full Strips and Arrays FAQ document.