The way of measuring Golgi pH seems to involve fusing a pH-sensitive GFP(pHluorin or deGFP4) with target protein(glycotransferases). Is there any other simpler method such as using a pH-sensitive GFP conjugated secondary antibody, so that regular ICC/IF could achieve this goal? Is there any commercial product available?
Hi Dannel,
Apart from great pioneering papers from Tsien lab for golgi pH measurement using the strategies of fusing a pH-sensitive GFP(pHluorin or deGFP4) with target protein(glycotransferases) another interesting paper was published in J Biol Chem, 1998 (see the attachment) exploiting the idea of GFP conjugated antibody. That might help you to solve the problem
Regards,
Hi Dannel,
I just came up another possible technique that can be used to measure golgi pH effectively. This technique utilized retrograde trafficking pattern of shiga toxin into the golgi compartments. Where the authors reported First, the recombinant B-subunit had been produced and labeled with either fluorescein isothiocyanate or DTAF (5-[4,6-dichlorotriazinyl]aminofluorescein). The resulting complex
retained its capacity to bind plasma membrane Gb3 receptors on select
cells and follow a retrograde pathway from plasma membrane to the Golgi complex.
Here, the dye resides for several hours without significant degradation and cells were then fluorescence imaged.
To me it seems to be a cool method.
I am sharing this article.
Good luck !!
Thank Amritlal Mandal for your very helpful information and the papers attached.
The first method you mentioned is quite suitable for the pH measurement of TGN, which might be limited to cis- or medial- golgi compartments. Whatever, it provides a new way of thinking for me.
The second method is so intriguing and I think it could solve my problem. Hoping a good result and thanks again!
Hi Dannel,
Glad to help you. Please give some feedback later once you are done with the expt.
Best-
To Amritlal Mandal,
It is so unfortunate to find that the method is highly cell line specific when preparing the experiment. Gb3 may not be expressed in CHO cells which is my target cell line; one paper has already reported that ("Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells"). It seems that only HeLa and Vero cells have been proved suitable in this method.
So it makes me a little bit sad to give up this brilliant method, seeking other appropriate ways to tackle this problem.
Still thank you for providing me two good methods which I don't know before.
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