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Co-immunoprecipitation (Co-IP) and Pulldown Assays

  • Confirm that the co-precipitated protein is obtained only with antibody against the target. Use monoclonal antibodies in the co-IP protocol. When only a polyclonal antibody is available, pre-treatment of the antibody with sample devoid of the primary target (bait protein) may be required to ensure that the polyclonal antibody does not contain clones or contaminants that bind prey protein(s) directly. Pre-adsorption to extracts devoid of target or pre-purification of polyclonal IP antibodies against an affinity column containing pure target antigen safeguards against a false positive co-IP.
  • In all pulldown assays, carefully designed control experiments are absolutely necessary for generating biologically significant results. A negative control consisting of a non-treated affinity support (minus bait protein sample, plus prey protein sample) helps to identify and eliminate false-positives caused by non-specific binding of proteins to the affinity support. The immobilized bait control (plus bait protein sample, minus prey protein sample) helps identify and eliminate false positives caused by non-specific binding of proteins to the tag of the bait protein. The immobilized bait control also serves as a positive control to verify that the affinity support is functional for capturing the tagged bait protein. 
  • Ensure that the antibody against the target antigen does not itself recognize the co-precipitated or pulled-down protein(s). Use independently derived antibodies that have demonstrated specificities against different epitopes on the target protein. Their use serves as verification that the target (bait)-directed antibodies have no affinity for the target-associated prey proteins recovered during the co-IP. Alternatively, an antibody against the co-precipitated or pulled down protein can be used to co-IP the same complex.
  • Determine if the interaction is direct or indirect and whether the interaction is mediated through a third-party protein that contacts both target and co-precipitated/pulled down protein. Immunological and other more sophisticated methods such as mass spectrometry may be necessary to answer this question.
  • Make sure that the interaction takes place in the cell and not as a consequence of cell lysis. Suggested approaches here involve co-localization studies and site-specific mutagenesis giving rise to mutants that perturb the binding process.
  • The tagged protein may have been degraded. Make sure that you included protease inhibitors in your lysis buffer.
  • Confirm that the fusion protein was properly cloned into the expression vector.
  • Use more lysate for the pulldown.
  • Use a more sensitive detection system such as SuperSignal™ West Femto Maximum Sensitivity Substrate (Cat. No.34095).
Yeast Two-Hybrid (Y2H) Screening

Here are possible causes and solutions:

Cause

Solution

Incorrect antibiotic used to select for transformants

Select for transformants on LB agar plates containing 10 μg/mL gentamicin (for bait plasmids) or 100 μg/mL ampicillin (for prey plasmids).

 

Didn’t use the suggested LR Clonase™ II enzyme mix or LR Clonase™ II enzyme mix was inactive

 

-Make sure to store the LR Clonase™ II enzyme mix at -20°C or -80°C.

-Do not freeze/thaw the LR Clonase™ II enzyme mix more than 10 times.

-Use the recommended amount of LR Clonase™ II enzyme mix

-Test another aliquot of the LRClonase™ II enzyme mix.

 

Not enough transformation mixture plated

Increase the amount of E. coli plated.

Here are possible causes and solutions:

Cause

Solution

Plates not replica cleaned

Replica clean immediately after replica plating, and again after 24 hours incubation.

 

Inadequate replica cleaning

Review Appendix, page 85 of the manual. Immediately after replica cleaning, plate should contain no remaining visible cells.

 

Too many cells transferred during replica plating

Review Appendix, page 85 of the manual. Transfer a minimal number of cells.

 

Incorrectly prepared 3AT plates

Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.

 

Incorrect incubation times

Incubate plates no longer than 60 hours (40-44 hours is usually best). Colonies arising after 60 hours are not likely to be of interest.

 

Here are possible causes and solutions:

Cause

Solution

Failure to add both bait and prey plasmids during transformation

Use bait and prey plasmids simultaneously in co-transformation procedures.

 

Incorrect selection plates

Plate co-transformations on SC-Leu-Trp plates.

 

Here are possible causes and solutions:

Cause

Solution

Candidate clones were false positives

Candidate clones could have been mutants of bait that self-activate. See page 50 of the manual for additional information on false positives.

 

Co-transformed pDEST™32 instead of bait plasmid

Retransform MaV203 with bait and prey plasmid.

 

Multiple prey clones in the original 3ATR transformants

Examine more ampicillin E. coli transformants for additional prey clones. Test each by reintroduction.

 

Here are possible causes and solutions:

Cause

Solution

Incorrectly prepared 3AT plates

Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.

 

Controls are too old or were mixed up

Return to the original DNA stocks provided, retransform on SC-Leu-Trp, and use fresh colonies.

 

Uneven replica plating

When replica plating, maintain an even pressure across the entire surface of the master and selection plates. Uneven pressure can result in the failure of cells to transfer.

 

Here are possible causes and solutions:

Cause

Solution

Incorrectly prepared 3AT plates

Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.

 

Strains being tested do not contain bait and prey

 

Confirm growth on SC-Leu-Trp plates.

Uneven replica plating

When replica plating, maintain an even pressure across the entire surface of the master and selection plates. Uneven pressure can result in the failure of cells to transfer.

 

Cause

Solution

Bait self-activates

Subclone segments of bait into pDEST™32 and retest.

 

Incorrectly prepared 3AT plates

Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.

 

Improper replica plating or replica cleaning

Review Appendix, page 85 of the manual. Immediately after replica cleaning, plate should contain no remaining visible cells (although a faint haze may be present on 3AT transformation plates).

 

Incorrect incubation times

Incubate plates no longer than 60 hours (40-44 hours is usually best). Colonies arising after 60 hours are not likely to be of interest.

 

Here are possible causes and solutions:

Cause

Solution

Gene of interest not in frame with GAL4 DNA Binding Domain encoding sequence

 

Sequence the DBD/test DNA junction.

Poor quality cDNA library

Determine the percent of vectors containing inserts and their average size.

 

Inadequate amount of cDNA library

 

Confirm concentration of library.

Test DNA cloned into pDEST™32 lacks or masks a domain required for protein-protein interaction

 

Clone and test alternative segments of the test DNA (bait)

cDNA library used does not contain proteins that interact with test protein X

-Screen a cDNA library from an alternative tissue, developmental time point, or organism.

-Determine whether the bait protein is expressed in the library.

 

Prey that interacts with bait may be toxic, unstable or require posttranslational modification

-Some posttranslational modifications cannot be accomplished in yeast.

-Make sure a cDNA library is constructed in pDEST™22 or pEXP-AD502 and not in other high-copy-number AD-vectors.

 

Bait may be toxic, unstable or require post-translational modification

-Some posttranslational modifications cannot be accomplished in yeast.

-Subclone segments of bait into pDEST™32 and retest.

 

Here are possible causes and solutions:

Cause

Solution

3AT concentration too low

Retest bait on various concentrations of 3AT.

 

Plates made incorrectly

Review Recipes on page 75 of the manual.

 

Improper replica cleaning

Review Appendix, page 85 of the manual. Immediately after replica cleaning, plate should contain no remaining visible cells (although a faint haze may be present on 3AT transformation plates).

 

Improper incubation times

Do not incubate plates longer than 60 hours. Colonies arising after 60 hours are not likely to be of interest.

 

Here are possible causes and solutions:

Cause

Solution

E. coli not sufficiently competent

Use ElectroMAX™ DH10B™ cells for library.

 

Too much DNA used

Use only 1 μl of DNA. Inhibitory compounds may reduce transformation efficiencies.

 

Incorrect selection or concentration

Select for plasmid on LB+ampicillin (100 μg/mL).

 

Alternative yeast DNA preparation procedure used

Use the method described on page 45 of the manual. Other procedures designed for high-copy-number vectors may not work with the ARS/CEN–based vectors used here.

 

DNA suspended in incorrect buffer

Electroporation is sensitive to ionic strength. Suspend DNA pellet in TE.

 

Crosslinking

The protein-protein interaction may be transient in nature. Often times, adding crosslinkers to capture putative interacting partners inside or outside of the cell may maintain the interaction through lysis and enrichment/purification. Membrane permeable crosslinkers like DSS (Cat. No. 21658) allow one to “freeze” interactions inside the cell, whereas membrane impermeable crosslinkers like BS3 (Cat. No. 21585) only work on the cell surface or outside the cell.

  • Choose more sophisticated crosslinkers with photo-reactive groups, which can be made to react at selected times and only in response to irradiation by UV light. Heterobifunctional crosslinkers with a thermoreactive group (spontaneously reactive) at one end and a photo-reactive group on the other end can be reacted first through the thermoreactive end with a protein that can be used as bait for other interacting proteins. The modified protein is introduced into a sample and allowed to interact with other proteins. Then the sample is exposed to UV light, which causes the photo-reactive end of the modified protein to covalently link to nearby molecules, thus “freezing” in place any interacting protein as a complex.
  • Remove non-reacted reagent by dialysis or desalting
  • Check to see whether there were any substances in the buffer that would prevent conjugation to the crosslinker. For example, primary amine containing buffers like Tris or glycine would out-compete amine-reactive crosslinkers like DSS or BS3. Sodium azide with a concentration over 0.02% is equally problematic.
  • Confirm that you used a membrane-permeable crosslinker for intracellular crosslinking.
  • Ensure that the pH was proper for the crosslinking to occur.
  • Use fresh crosslinkers.
  • For a photo-reactive crosslinker, check that the sample was exposed to the proper UV wavelength (300-370 nm) at the proper distance and exposure time.

Label Transfer

  1. Determine the label incorporation of the bait protein before photoactivation with the prey protein, by western blot analysis.
  2. Check the UV light source to ensure good photoactivation (see Photolysis Information section of the Sulfo-SBED manual for guidelines).
  3. If the target (prey) protein concentration is too low, enrich the protein system if possible.
  4. Optimize the antibody concentrations used in western blot.
  5. It is possible that the protein interaction is blocking the epitope, which may occur with monoclonal antibodies and peptide-directed antibodies; in this case, use an antibody from a different source or probe with Streptavidin-HRP.
  6. Milk contains endogenous biotin and its use as a blocking buffer during the detection assay could mask the bands of the biotin-labeled interaction partner.

There are several approaches to eliminate excess bands on a blot. These include the following:

  1. Try a different blocking agent (keep in mind that milk contains high amount of biotin and could mask any specific results).
  2. The target sample may contain other proteins which bind to the labeled ligand. Add Tween™-20 (0.005%, v/v) to the 1X Label Transfer Buffer. Alternatively, increase the stringency of the washes by increasing the concentration of detergent from 0.05% Tween-20 to 0.1%, increase the salt content of the wash buffer, or adjust the pH of the buffer to see if any of these steps will release the non-covalently bound proteins to the target protein.
  3. Enrich the complex sample using immobilized monomeric avidin.
  4. It is possible that too much protein was loaded on the electrophoresis gel; in this case, apply a smaller amount of protein on the gel.
  5. Reduce the amount of sulfo-SBED biotin used to label the bait protein as the crosslinking process could be covalently attaching the bait protein to non-relevant proximal proteins.

Confirm that the reducing agent is fully cleaving the bait protein off of the target protein. Increase the concentration of the reducing agent or use fresh reducing agent if the bait:target protein is not being cleaved apart. 

Far-Western Analysis

The following are the reasons and remedies for low signal in far-western blot application:

  1. Streptavidin-HRP is too little or has low activity: Try 2- to 5-fold higher concentration or use fresh streptavidin-HRP.
  2. Biotinylated-protein probe is too dilute: Try 2- to 5-fold higher biotinylated-protein probe concentration. 
    Note: Alter the biotinylated “probe” (bait) protein concentration only if adjusting the Streptavidin-HRP concentration has no effect or results in high background. 
  3. Probe protein not sufficiently biotinylated: Biotinylate probe (bait) protein again with a higher molar excess of the biotinylation reagent to the protein concentration; mole-to-mole ratio of biotin may be determined using HABA (Cat. No. 28010) or Fluorescence Biotin Quantitation Kit (Cat. No. 46610) each time the probe is biotinylated to make sure that consistent biotinylation level is obtained.
    Note: There can be a fine line between sufficient labeling for detection and over labeling with subsequent steric hindrance and loss of signal. Optimizing the labeling reaction prior to the far-Western can reduce downstream optimization, which can use up precious samples.
  4. Biotinylated protein probe interacts weakly with “prey” protein: Use native conditions to separate samples, as denaturing conditions may alter the proteins such that they do not interact with the “probe” during electrophoresis and transfer.
  5. It is possible that a change in three-dimensional structure took place due to the protein absorbing into the membrane, changing the interaction site. In this case, far-western analysis may not be an appropriate approach for detection of a protein-protein interaction and cross-linking or label transfer may be other viable options to confirm the interacting partnership. 

There are several approaches to eliminate excess bands on a blot. These include the following:

  • Use a compatible blocking buffer to dilute the biotinylated-protein probe and Streptavidin-HRP (avoid milk which contains endogenous biotin and can interfere with signal production).
  • Target sample may contain other proteins which bind to labeled ligand. Add Tween™-20 (0.005%, v/v) to the 1X Label Transfer Buffer. Alternatively, increase the stringency of the washes by increasing the concentration of detergent from 0.05% Tween-20 to 0.1%, increase the salt content of the wash buffer, or adjust the pH of the buffer to see if any of these steps will release the non-covalently bound proteins to the target protein.
  • Enrich complex sample using immobilized monomeric avidin.
  • It is possible that too much protein was loaded on the electrophoresis gel; in this case, apply a smaller amount of protein on the gel.