Hello Lyra,

This is puzzling. FAM typically does suffer some decrease in fluorescence with lower pH. This is probably a trivial suggestion, but I would suggest that you co-dialize the protein and the fluorescent peptide in the same buffer in order to remove the pH dependence. All the best!

Akash

Hey Akash,

Thank you for your suggestion! I will try it out~ 

Good to see you here :-)

Best,

Lyra

You should have been using a buffer to control the pH, so the isoelectric points of the protein and peptide should not have affected the pH. For fluorescein, the pH should be kept at 7 or above, because fluorescein loses fluorescence below pH 6. If a large change in pH from below 6 to above 7 was occurring in your experiment when the protein was added, that would explain the fluorescence increase.

If a pH shift was not responsible, another plausible reason for the large fluorescence enhancement is that the fluorescence of the fluorophore on the peptide was substantially quenched by an interaction with the peptide, and this quenching was relieved when the peptide bound to the protein. If this is what happened, then you can use the effect as a binding measurement, rather than polarization.

A good control experiment would be to show that the fluorescence increase is reversed when an unlabeled version of the same peptide is added.

Hi Adam,

I have been using a 50 mM HEPES buffer (pH = 7.5), 150 mM NaCl for my experiment. So I think the pH should be quite stable.

I really like your suggestion of using the control experiment to find out whether the fluorescence increase is due to the fluorophore binding to the peptide & when peptide bind to protein, this quenching is released. 

My peptide suppose to have strong positive charge in my buffer. Have you seen this kind of binding between positive-charged peptide & FAM?

Thank you so much for your help!

Have a great weekend!  

Lyra 

I saw quenching of fluorescein attached to a DNA oligonucleotide when a complementary unlabeled strand was added.

Fluorescein has a negative charge, so it is plausible that it could suffer static quenching due to an electrostatic interaction with positive charges on a peptide to which it is attached.

Thank you again Adam! I will let you know how the experiment goes.

Have a great day,

Lyra

There is also possibility that your probe stacks to the surfaces and the actual concentration in solution is lower. When you add the protein then binding helps to remove the probe from the surfaces and you detect the increase of the signal. We have seen similar things a number of times. Adding of surfactants or using of low-protein binding plates have helped, but not always completely.

Hi Erki,

Thank you for your suggestion!

I will try that also~ 

Best,

Lyra

I agree with Erki that prevention of probe binding to the surface of assay plates is desirable. For me, it is standard practice to include a sub-CMC concentration of non-ionic detergent, such as 0.01% Triton X-100, in the buffer to minimize binding of fluorophores and proteins to polystyrene plates.

HI Lyra,

I agree strongly with Adam for the quenching state of your FAM-conjugated peptide... I will also add that it depends too on your peptide length and contains of aromatic amino acids such tryptophan or phenylalanine ... if so, they are likely to quench fluorescence from Fluorescein dye ... I will say also it depend on your working concentration ... if you are working at high concentration of your peptide it does not help you ... especially if your peptide interacts with each other... (aggregate) that will produce quenching wich is lowered by your added protein interaction with your peptide after that.

If it s true you can start with a fluorescence standard curve using diff peptide-FAM probes concentration and determine the good concentration for the best fluorescent signal while adding fixed protein concentration or not in your PH controlled solution.

I had the same issue in the past when i was trying to synthesize my home made peptide fluorescent probe... Finally, don t forget to setup your reader at the right properties ( especially for the well plate depth reading position, same volume in each wells etc... lot of people forget to calibrate their reader before the use)

All the best

Thank you Adam & Foued!

Here comes some update. Today I tried a different buffer that is previously used for the FP assay of this peptide (50 mM Tris 50 mM NaCl 5% glycerol). I thought the 5% glycerol will do the trick. But no luck! I am going to try adding 0.01% Triton X-100 next.

I am also testing if the peptide is indeed self quenching by competing with unlabeled peptide. Unfortunately, I only see a slight drop of fluorescent signal (~9500 --> ~8500) after 1 hour incubation. I am going to read 30 mins later but I am not too confident about this.

I am currently testing 4 different FAM peptide concentrations: 10, 5, 2.5, 1.25 nM. Do you think the concentration is reasonable or should I go higher.

Foued, could you share with me some good references to help guide my adjustment of FP plate reader? Or should I just read the manual for our plate reader?

Finally, have anyone ordered peptides from Genscript? I wonder if the problem lies within the peptide quality?

Thank you all again & I will keep updating of my results.

Best,

Lyra

Another update is that my FAM-peptide seems to aggregate after freezing in -80. I dissolve it in PBS. Any trick to reduce the aggregation after thawing the aliquot?

Warm it ! After total defreezing at room T or 4 C .. You can warm your solution for few minute i d say at more than 65 C ... I used to warm my probe at 95 C for 2min30 second without any degradation to avoid dimerisation and worked ..

Best

Thanks a lot, Foued!

Have a great day~

Lyra

Some more update:

I realized today that I used a wrong case of polystyrene 384 plates that has high binding surface for my earlier experiments. I thought I found the root of my problem.

Therefore, today I used a different kind of black flat bottom 384 well plate that has normal surface for another run of FP assay, I even used FAM labeled compound to adjust the G value of the plate reader. Still! Same problem is observed! Although the fluorescent increase become ~2-3 fold. The FP signal decreased when I add my protein! So frustrating!

Any suggestion will be deeply appreciated!

Also, can anyone recommend good 384 well black plate for FP assay?

Thank you in advance!

Lyra

When using polystyrene plates, it is advisable to include an agent in the buffer to block the hydrophobic surface from binding the assay components. My practice is always to include a nonionic detergent at a concentration below its micellar concentration. An example is 0.01% Triton X-100.

Hi Adam,

Adding Triton X100 will be the next thing I try tomorrow. I wonder have you ever seen FAM-peptide+protein has lower fluorescent polarization signal than peptide only control? I wonder what that means?

Thanks a lot for your help!

Best,

Lyra

Hi Lyra,

Here what I think... 

Fluorescence polarization increases as molecular weight increases.

Fluorescence polarization increases as solvent viscosity increases.

Fluorescence polarization decreases as the excited state lifetime of the dye (τ) increases. When ur ligand binds to your protein, it increases its effective volume and therefore tumbles more slowly, causing more emitted light to remain polarized in the same plane as the excitation light;  therefore your FP increases..

Somehow, could the possible response is that your peptide does not effectively bind to your protein ??!  Or you may have fluorescein interfering with your binding sites... BODIPY dyes generally produce less perturbation of receptor-binding affinity and other activity parameters than conventional dyes such as fluorescein and rhodamine. Or you are doing measurement before your reaction reach equilibrium state ?? Or your peptide concentration in the solution is too high ... you may need to adjust your parameters.. 

Here a good ref for troubleshooting for Fluo-polarization assay: Analysis of protein-ligand interactions by fluorescence polarization. Ana M Rossi & Colin W Taylor

Nature Protocols 6, 365–387 (2011) doi:10.1038/nprot.2011.305

I hope will help you !

Best

Hi Foued,

Thank you so much for your suggestions!

I covered up the assay plate I prepared yesterday and read it again this morning. I saw that the wells with protein+peptide now has 10 x more fluorescence than just peptide alone. For example, in the well with 5 nM of FAM-peptide, I saw that I have 30000 AU perpendicular fluorescence in wells with both peptide and 20 uM of protein while only have 3000 AU in peptide only control. Similarly I have 27279 AU in parallel fluorescne in peptide+protein well while having 4000 AU in peptide only well. Yesterday the difference between peptide+protein vs. peptide only well is only 2-3 fold. But after overnight incubation, the difference become much bigger. 

But same trend hold true, I still have higher FP signal in peptide only plate. Strange!

I am going to try following strategies today: 

1. Adding 0.01% triton X-100 to my buffer

2. Increase peptide concentration to 100 nM

3. Use different 384 well plates

BTW, wells with just buffer alone (without FAM-peptide) has very strong FP signal. Is that normal? Does the plate suppose to have intrinsic fluorescence? I used the OptiPlate 384-F. Have you used that before? 

I thought this is just a simple & straight forward assay but it seems to more difficult than I thought! 

I will read though the nature protocol paper you sent me.

Thanks again for your help!

Best,

Lyra

Hi All,

I have tried following parameters:

1. Adding 0.01% Triton X-100 to my buffer

2. Increase peptide concentration to 100 nM

3. Use different 384 well plates (5 different kind of black plates)

Simply put, nothing works!!!

0.01% triton X-100 can reduce the FP signal difference between "peptide only" and "peptide + protein". However, adding protein to FAM-peptide actually reduce the FP signal. We tried with other FAM-labeled peptide and another target, it works. Therefore, there is something about this peptide that prevent it from giving normal FP response.

My theory is that the peptide aggregate on itself and therefore has high FP signal (in aggregate). Adding protein prevent the aggregate formation and therefore reduce FP signal.

Next I am going to try following strategies:

1. Including 1mg/ml BSA & 0.01% Triton X-100 in the solution

2. Use intrinsic tryptophan fluorescence on the peptide to track protein binding (I just want to know if it binds or not to my target)

Here is my update & any thoughts, suggestions will be deeply welcomed.

Best,

Lyra

Update: 1mg/ml BSA doesn't change the FP signal much. Adding protein still slightly decrease the FP signal (~70 --> 35).

I am going to use ThermoFluor to measure peptide binding tomorrow (using biotinylated version of peptide).

Best,

Lyra

Hi Lyra,

I am happy to see that you got a positive control experiment where you see that your protocols worked with another peptide + another target protein)... This is however strongly show that your peptide is not that specific to bind with your target ... I think after your great thought using intrinsic tryptophan fluorescence will conirfm it ... Maybe you should find another peptide ?? is this a comercial peptide or your proper custum design peptide ?

Best

Hi Foued,

This peptide is ordered through GenScript and there was a publication using same peptide for FP assay (but they synthesized themselves) and it suppose to work.

Yesterday, we increase the protein concentration to 100 uM and still cannot see any binding. I am currently doing ThermoFluor experiment to determine the binding affinity of peptide (biotinylated version) to protein. Will let you know how that goes.

We also suspect that the affinity is a bit lower than what's reported. 

Best,

Lyra

Here comes latest update!

I have done ThermoFluor with the protein target + biotinylated peptide today.

It turns out that the peptide only start to stabilize the protein at ~60 uM. This suggest that the previous reported 10 uM Kd probably is off quite a bit.

Oh well, at least I proved that the peptide binds, just that the affinity is WAY too low for FP assay to work.

Thank you all for your help!

Best,

Lyra

Hi Lyra,

Really happy that you figured out :-)... most of the time reported data in publication are not reproducible at same level btween labs so curful with conclusions :-)

Hope you will find a great affinity peptide for your experiment.

Best

I've seen examples where peptide binding affinity was strongly affected by the conditions, particularly the salt concentration. If you think charge interactions may be involved in peptide recognition, try lowering the salt concentration.