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.

Download the full Strips and Arrays Protocol here:

For use with product numbers: P-6001, P-6100, P-6002, P-6003, S-6000, S-6001, P-6111, and P-P100

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).

PIP Strip - Blocking Buffer Optimization

Buffers

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

Wash SolutionsBlock Solutions
TBS tablet (Amresco K859) in 100 mL dH2OTBS or PBS + 3% BSA: add 3 g fatty acid free BSA to 100 mL TBS or PBS
For TBS-T, add 0.1% v/v Tween-20For PBS-T + 3% BSA: add 3 g fatty acid free BSA to 100 mL PBS-T
PBS table (Sigma P4417) in 200 mL dH2OTBS or PBS + 1% milk: add 1 g non-fat dry milk to 100 mL TBS or PBS
For PBS-T, add 0.1% v/v Tween-20TBS or PBS + 0.1% ovalbumin: add 0.1 g ovalbumin to 100 mL TBS or PBS

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

DescriptionCatalog NumberConcentrationBlock SolutionSecondary Antibody
Multi-PIP Grip (LL5α) G-9901 0.5 μg/mL PBS-T 3% BSA K-SEC2
Multi-PIP Grip (LL5α) G-99012.0 μg/mL TBS-T 3% BSA K-SEC2
PI(3)P Grip (p40PX) G-0302 1.0 μg/mL PBS-T 3% BSA K-SEC2
PI(4)P Grip (SidC_3C) G-0402 0.5 μg/mL PBS-T 3% BSA K-SEC2
PI(4,5)P2 Grip (PLCδ1) G-4501 0.5 μg/mL PBS-T 3% BSA or PBS 1% Milk K-SEC2
PI(3,4,5)P3 Grip (Grp1) G-3901 0.25 μg/mL PBS-T 3% BSA or PBS 1% Milk K-SEC2
Anti-PI(4)P IgM Z-P004 1.0 μg/mL PBS-T 3% BSA Anti-mouse IgM HRP
Anti-PI(3,4)P2 IgG Z-P034 2.0 μg/mL PBS-T 3% BSA K-SEC1
Anti-PI(3,4,5)P3 IgG Z-P345b 4.0 μg/mL PBS-T 3% BSA K-SEC1

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.
We do not recommend the use of cell lysates with these products as it contributes to high background and can obscure positive signals due to non-specific binding of other cellular components or proteins.
A complete absence of signal (white membrane) is typically due to loss of activity of the binding protein or high detergent concentrations in the assay buffer. In either case, it is recommended to run a positive control for the lipid of interest in order to determine the issue. If aberrant binding is observed this may also be due to harsh detergent conditions or over development of the membrane after application of the detection reagent. Additional details can be found above in our full FAQ document.
All strips and arrays have been internally validated using HRP-conjugated secondary antibodies and can be used with precipitating reagents or chemiluminescent solutions. Use of fluorescently conjugated secondary antibodies is also possible with these products but we encourage the user to optimize the antibody concentration in order to decrease background signal.
High background is most likely attributable to insufficient washing or excess binding protein used in the initial incubation step. Background signal may also be reduced by adjusting the composition of the blocking buffer.
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