Protein Labeling, Crosslinking, and Modification Support—Troubleshooting

See our recommendations below:

• Crosslinkers are typically shipped as solids and most are labile to moisture. Upon receipt, store as recommended. Resolubilze the crosslinker shortly before use. If using a photoreactive crosslinker, protect from light during storage and handling.
• If the proteins are intracellular, make certain that the crosslinker is cell permeable.
• The reactive sites on both proteins must be on or near the surfaces of the proteins and accessible. If uncertain about the accessibility or location of the reactive sites on the proteins, consider using a non-specific photoreactive crosslinker.
• Ideally the crosslinker should be added to cells in a simple buffer to avoid reaction of the crosslinker to media components. Make certain the buffer components do not include ingredients that can react with the crosslinker. For example, amine-reactive crosslinkers should not be added to buffers containing Tris or glycine. EDAC and other carbodiimide-based crosslinkers should not be added to buffers that contain amines (Tris, glycine, etc.), phosphate or carboxylated compounds (e.g. citrate buffer). 
• If using a zero length or short crosslinker, try a longer linker. 
• Use different concentrations of the crosslinker per a defined number of cells and cell density. Too low a cell density promotes hydrolysis of water-labile reagents and too high a cell number or density may result in not enough crosslinker. 
• During the initial extraction/purification of the proteins, be certain to include protease inhibitors. Flash freezing and working through the lysis quickly also ensures the integrity of the sample.
• It may be necessary to know something of the half-life of the protein in the cell. Rapid turnover of the proteins of interest may require careful timing from induction to crosslinking and on to lysis. 

Yes, the reaction may be performed in acetonitrile. An example protocol can be found here.

The EZ-Link™ kit utilizes sulfo NHS-SS-biotin that contains a disulfide bond which can be cleaved with a reducing agent. However, if you do not want to use a reducing agent after pulling down the biotinylated protein with streptavidin or NeutrAvidin™ resin, you can boil the resin in non-reducing SDS-sample buffer. This treatment will dissociate biotin from streptavidin or NeutrAvidin™ resin by the denaturation of the streptavidin/NeutrAvidin™ resin. 

To limit non-specific binding, first pre-clear your sample using the base agarose resin and then run the precleared sample on the resin coupled to the small molecule. We offer Control Agarose Resin (Cat No. 26150) for this purpose. 

Example procedure to pre-clear lysates, or to use with the serum, before using sample for IP is shown below:

1. For 1 mg of lysate , add 80 μL of the Control Agarose Resin slurry (40 μL of settled resin) into a spin column. 
2. Centrifuge column to remove storage buffer. 
3.  Add 100 μL of 1X Coupling Buffer to the column (alternative buffer: PBS), centrifuge and discard the flow-through. 
4. Add 1 mg of lysate to the column containing the resin and incubate at 4ºC for 30 minutes to 1 hour with gentle end-over-end mixing. 
5. Centrifuge column at 1000 × g for 1 minute. Discard the column containing the resin and save the flow-through, which will be added to the immobilized antibody

BS3 is a water-soluble crosslinker but it is not membrane permeable. DSS is the membrane-permeable alternative if the aim is to perform intracellular crosslinking. Either the BS3 or DSS solution should be added at a final concentration of 1-5 mM. Sufficient volume used should be enough to cover the surface of the cells: see the link below for volumes typically used for cell culture media. For example, use about 2 mL of media for a 6-well plate well. Use the same volume for the crosslinker. Refer to our chart for useful information of various sizes of cell culture dishes and flasks.

After incubating the crosslinker solution, you do not need to aspirate it. Instead add the Tris quenching buffer to a final concentration of 10–20 mM Tris. Based on the stock concentration of your Tris quenching buffer, you can determine the volume need to get the 10–20 mM final concentration.

Unfortunately, we do not have a carboxyl-reactive crosslinker that provides a spacer arm to give distance between the two molecules to be linked. We do offer amine/sulfhydryl reactive crosslinkers that are available with various lengths of spacer arms. 

To provide a linker between the beads and the target protein, you would need a molecule with a primary amine (to conjugate to the carboxyl group on the beads with EDC) on one end and another reactive group on the other (to conjugate to the protein). For instance, sulfo-SMCC (Cat. No. 22622) could be used in conjunction with PDPH (Cat. No. 22301):

The method is outlined below:
1. Conjugate the target protein to the NHS ester (amine-reactive) group of Sulfo-SMCC. This will produce a maleimide-activated target protein via its primary amines (lysines and N-terminus).
2. Remove the excess sulfo-SMCC by desalting or dialysis.
3. React the maleimide-activated target protein to PDPH via sulfhydryls. (Lysines and N-terminus now are connected to hydrazide (amine) group with an extended linker)
4. Remove excess PDPH by desalting or dialysis.
5. React the hydrazide-activated target protein with carboxyl beads in the presence of EDC.
6. Use appropriate buffer to rinse off excess target protein from the carboxyl beads. 

The spacer arm length (12.0Å) of our DSP (Cat. No. 22586) and other products was determined using a molecular modeling tool, such as ChemDraw. 

There are two reactions occurring when a molecule is conjugated to CarboxyLink™ Resin in the presence of EDC. These reactions may occur simultaneously (at pH 7.0-7.2) or separately (moving from an acidic pH to a slightly alkaline pH). Adding sulfo-NHS to the reaction along with EDC helps increase the yield of the reaction.

The activation reaction of the molecule’s carboxyls with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2; however, the reaction of NHS-activated or sulfo-NHS-activated molecules with primary amines is most efficient at pH 7-8. For best results, perform the first reaction in MES buffer (or other non-amine, non-carboxylate buffer) at pH 4.7-6, then raise the pH to 7.2-7.5 with phosphate buffer (or other non-amine buffer) immediately before reaction to the amine-containing molecule. The reaction should be done with EDC that is freshly prepared. 

The cysteine sulfhydryl is very labile when exposed to air or in solution, and it can easily oxidize with another sulfhydryl. This typically happens between sulfhydryls from two molecules, but it can sometimes occur if there are at least two cysteines present on the same peptide or protein. This oxidation forms a disulfide bond and changes the two cysteines into cystine.

TCEP is one of three commonly used reducing agents that can break a disulfide bond. The other common reducing agents are beta-mercaptoethanol (β-ME or 2-ME) and dithiothreitol (DTT).

In step A-3 of the manual, one would carry out the TCEP reduction by adding 0.1 mL TCEP to the solution prepared in step A-1 and let incubate for 30 minutes. After the SulfoLink™ Resin is prepared in steps B-1 to B-3, one would add the sulfhydryl-containing molecule (result of step A-3) to the SulfoLink™ Resin.

Step A-3 refers to “Equilibrate the SulfoLink™ Column during this incubation step.” This equilibration procedure is laid out in steps B-1 to B-3. Essentially, you are removing the storage solution from the SulfoLink™ Resin; then washing the SulfoLink™ Resin with Coupling Buffer.

To “Equilibrate column with 6 mL of Binding/Wash Buffer” after removing storage solution: Add 6 mL of Binding/Wash Buffer to the column containing the peptide-bound SulfoLink™ Resin. Centrifuge at 1,000 x g to remove the Binding/Wash Buffer. Perform this washing (add 6 mL of Binding/Wash to the column and centrifuge at 1,000 x g) two more times. There is an option discussed in the note above the “Materials Required” section that a traditional gravity-flow method can be performed in lieu of spin-purification. This means that you can allow liquid to drip through the column by gravity in lieu of centrifuging the buffer through column. 

If the unmodified small molecule precipitates by itself (i.e., when diluted with PBS only), it is not very soluble in aqueous solutions. You can try increasing DMSO to the buffer to about 20%. If this does not help in solubilizing the precipitant, most likely this is the crosslinker-activated molecule. We would recommend titrating the amount of organic solvent (DMSO) in the buffer system to maintain the small molecule in solution but still have a buffer system that can be used for any further reactions. 

To confirm that the bead to antibody crosslinking was successful using BS3 (bis(sulfosuccinimidyl)suberate), after quenching the antibody-conjugated bead, compare the following fractions:

1. Free antibody (prior to conjugation) as a positive control
2. After conjugation, add wash buffer to beads and see what, if anything, did not bind
3. Then add elution buffer, which is typically 100 mM glycine, pH 2.0-2.8, to the beads to see whether anything comes off

It is easiest to track these fractions on the gel to see whether any antibody came off your resin. Absorbance might be thrown off by presence of the glycine in your elution buffer resulting in a false positive. Finally, make sure to rinse your beads with wash buffer as soon as you can because extended periods at low pH can damage your antibody.

EZ-Link™ TFPA-PEG3-Biotin (Cat. No. 21303) can be used in a wide variety of buffers. However, acidic pH and reducing conditions inactivate the aryl azide. We are unaware of any other photoreactive biotinylation reagents that could be used at low pH.

The crosslinker may have hydrolyzed during storage. If an alternative method in the instructions recommends DMF or DMSO, use DMF or DMSO for initial dissolving. You can dissolve the crosslinker at higher concentrations in DMF or DMSO and then as the working concentration should permit diluting 100-fold so there would be very little solvent left in the final reaction.

To keep the reagent stable as long as possible we have the following suggestions: 

1. Keep exposure to the atmosphere to a minimum.
2. Equilibrate to room temp fully before opening the cap. This prevents any condensation forming inside the vial.
3. Reseal under a stream of nitrogen or other inert gas.
4. Store at 4˚C in a sealed container with dessicant.

Yes, this is possible with sulfo-SDAD and sulfo-SBED, but not with SPB. When using any of these 3 linkers, a purified protein should be labeled first using the NHS end of the linkers; this should be done in the dark. After labeling the purified protein with the photoreactive linker, you should add the DNA in excess relative to the amount of protein, allow sufficient time for binding and then, expose this to light. 

The first choice would be the SPB followed by the sulfo-SDAD followed by the sulfo-SBED. With SPB the psoralen is very specific for DNA, such that after the protein has interacted with the DNA the psoralen should link them together efficiently. With sulfo-SDAD the diazerine is not specific towards DNA—it will pretty much react with anything so there is more risk in the formation of protein multimers. Sulfo-SBED works well, but involves biotin transfer rather than actual linking. Interacting molecules are less obvious from background.

If the crosslinker is crossing the membrane, it probably is somewhat dose-dependent. After washing the cells after crosslinking, there would not be much BM(PEG)3 available to further crosslink the proteins after lysis. So either the linker is crossing the membrane or the cells are already compromised at the time of labeling. To quench this crosslinker, use cysteine as it does not have significant reducing activity.

Overall, NHS ester groups are much more reactive than maleimides, and there will likely be multiple lysine targets compared to only one cysteine, if you cannot get the BM(PEG)3 working, we recommend using the BS(PEG)5 (Cat. No. 21581).

We have no evidence that HPDP-biotin (or any pyridyldithiol compound) reacts with anything other than sulfhydryl (-SH) groups. However, many sulfur-containing compounds interconvert among several different forms. For thiouracil, with the different tautomers that are possible, the =S is in equilibrium with –SH, which can then react with the HPDP.

To use the L-photo-leucine, you will need to use a medium that is free of L-leucine. The 293 medium has all essential amino acids so they will compete with the photo amino acids for incorporation into your proteins. Our product DMEM-LM (Cat. No. 30030) is deficient in L-leucine and L-methionine.

Check that the appropriate molar excess of reagent was used for the reaction. The addition of too many labels or over-crosslinking the protein will affect the net charge and thus change the proteins pI and solubility. 

Many crosslinking and labeling reagents are moisture sensitive and must be prepared immediately before use. Due to the nature of the reagent, stock solutions cannot be stored. 

First, ensure that the target functional group is available on the molecule of interest. Some groups may not be available and so a reagent which targets a different functional group should be used. Second, ensure that no interfering proteins or reagents are present in your buffers (e.g. Tris or glycine buffers are incompatible with amine based reactions). Lastly, check that a sufficient amount of reagent was added to the reaction, the molar excess of reagent to protein may need to be optimized empirically. 

Hydrolysis as well as conjugation releases NHS as a leaving group that absorbs strongly at 260-280 nm (λmax = 260 nm; ε = 9700 M^-1cm^-1 in NH4OH). Therefore, by comparing the absorbance before and after intentional hydrolysis with strong base, one can assess the reactivity remaining in the original sample of NHS ester biotinylation or crosslinking reagent. Please see this Tech Tip for details of the procedure.

Excess reagent can be easily removed through dialysis or desalting methods.