Hi Zhiyuan,

I just discover the MST assay. Looks promising!

If no conditions which preserve an active form have been described so far, i think you have to try other detergents... GPCR function is very sensitive and a rigorous screening is needed in order to preserve it.

Are you sure that you GPCR-GFP is functional in the cell before the lysis (effect of the fusion/level of expression)?

Based on the paper of seidel et al, 2013, they used purified and E.coli expressed receptors. The purification steps need the add of some phospholipids to preserve the receptor function.... Maybe you can think to use a tag receptor and purify it in a similar way from the cell lysate.

Best

Guillaume

Hi Guillaume，

The GFP-GPCR is functional is the overexpressing HeLa cell. Due to lack of tag used for purification, we just want to do MST using cell lysate supernatant to detect the interaction with the small molecules. The  obstacle we confronted is the activity of the GFP-GPCR. Now some detergent such as OG, DDM,DM,LDAO and DPC are available to be tested. But I just want to know how to use the detergents. Should I add the detergents to PBS  or lysis buffer such as MPER plus protease inhibitor? Could  anyone give me some advice? 

Thanks a lot!

Dear Zhiyuan,

Ok, here the limit of my expertise...

I did two types of experiments with GPCR.

i) binding test with membrane preparation. In this case, we worked on quite big cell membrane fragments with many receptors on it but still functional (no detergents added). Can you work with such type of preparation?

ii)Western blot on solubilized receptors. In the case we added DMM 1% to the membrane preparation. We optimized both the detergent and its concentration in order to have an over representation of one receptor per micelle. Nevertheless, by this treatment, function was impaired quite significantly...

Hope that will help you.

Best

Guillaume

Dear Guillaume，

Thanks for your advice。

Firstly， I cannot do theexperiment”i) binding test with membrane preparation“.

Do you have some protocol for reference?

Secondly, I indeed do MST using such method. That is to say, lysing the cells to collect the supernatant and using the supernatant to perform the MST assay. Do you mean that however optimizing the detergent concentration or types will not get fucntional GPCRs?

Best 

Zhiyuan

Dear Zhiyuan,

Please find a current protocol for membrane preparation in the PlosOne article (2012). It works very well for muscarinic, adrenergic and dopaminergic receptors.

Maybe you can check for similar protocol for a receptor more closer to yours. Membrane preparation is something quite common.

You can get a functional GPCR with the use of detergents/phospholipids. But in my mind, such task need a lot of optimization (and luck, maybe). This is useful if you need a solubilized receptor in your assay. This, I don't know. You have to tell.

Hope it's help...

Guillaume

Article Engineering of Three-Finger Fold Toxins Creates Ligands with...

Dear Guillaume，

Thanks for your help

Best

Zhiyuan

the softest detergent you could use is digitonin, otherwise you could try DDM or LMNG I would solubilise at around 1% detergent with membranes at around 8-10mg/ml.  Do not use detergents such as triton or NP these are good detergents for molecular biology but not for GPCR biochemistry.  Before running your assay I would also check the quality of you expression by FSEC (as you have a GFP) this will give you a crude idea regarding the quality of your solubilised material

Hi James,

Thanks for your advice.

In your case, does it mean that I only add detergents such as DDM or LMNG in the lysis buffer? Could you mind sharing your lysis buffer recipe for GPCR biochemistry.

And how can you quantify the concentration of membrane? Is there any reference or protocol for such experiment?

Thanks a lot in advance.

Best

Zhiyuan

In your case, does it mean that I only add detergents such as DDM or LMNG in the lysis buffer? Could you mind sharing your lysis buffer recipe for GPCR biochemistry

I would add the detergent (I would start with digitonin as this is the mildest detergent but use a good quality one such as Calbiochem) to your current buffer (without he Triton or NP) every receptor is different and each one will take optimisation of its buffer for example the amount of salt (sodium can act as a allosteric modulator) or divalent (Mg or Zn etc).  You may need to play around with the amount of detergent to optimise your assay but always remember to keep the amount of detergent above the CMC (and you will need significantly more for solubilisation)

And how can you quantify the concentration of membrane? Is there any reference or protocol for such experiment? 

We use a simple BCA assay we harvest cells, wash with PBS then resuspend homogenise then spin in ultra and collect membranes (you can introduce a low speed spin prior to ultra to pull down large stuff)

Thanks James 

Hi Zhiyuan,

Another possibility that you could not identify the interaction between GPCR and the compound might due to the unspecific binding. There might be other molecules in cell lysate, which also binds to the small compound.

Have you ever thought about to purify the protein firstly, such as His-tagged or flag-tagged to the GFP-fusion protein, then perform MST experiment? There is a great publication on Nature article 2014, Parker and Newstead identified the interaction of a 12-helices transmembran nitrate transporter with nitrate by using MicroScale Thermophoresis (see attachment). Hopefully it will be helpful for you.

I am an application specialist of MST at NanoTemper Technologies. If you don’t mind, could you let me know your email address? I can contact you privately and I would like to give you more support for the .ntp data analysis.

Best

Zhuo

Hi Zhuo,

Sorry to answering you too late.Glad to hear that you will provide help.

My E-mail: [email protected]

Thanks a lot!

sincerely

Zhiyuan

Hey Zhiyuan, 

We have just started to optimise cell lysis for extracting functional GPCR in cell lysate to carry out MST. I was wondering if you could share what worked for you in the end? 

Thanks!

Deeptha