McClean:Phluorin Calibration

Overview

Ratiometric pHluorin is a pH-sensitive GFP derivative. See Miesenbock, et al 1998 for the original description of this protein. Wild-type green fluorescent protein (GFP) exists in two conformations and therefore has a bimodal excitation spectrum with peaks at 395nm and 475nm. The authors took advantage of these conformation states, and with some clever amino-acid substitutions facilitated pH-dependent switching between the states. Thus, the relative emission of pHluorin when excited at 395nm vs 475nm is dependent on pH.

This protocol describes how to measure a calibration curve for pHluorin in your yeast strain of interest. The relative emission at 395nm or 475nm excitation is measured for permeabilized cells in buffers over a range of pH. This protocol was adapted from Orij 2009. See their paper (Figure 2a) for a good example of what your calibration curve should look like.

Materials

• Strain of interest
• Low Fluorescence Media with appropriate amino acids for your strain
• Digitonin (Acros Organics, #407565000)
• Phosphate Buffered Saline, pH 7
• Buffers (see below)

Buffers

We use a citric acid/Na₂HPO₄ buffer to create pH values ranging from approximately pH 5 to pH 9. Tina Hansen made the following convenient chart when she tested this protocol. You should probably test the pH of your buffers (as Tina did) to make sure that you know what the actual pH is (instead of relying on what it "should" be based on a buffer table).

pH actual	Label on tube (pH expected)	x ml 0.1M-citric acid (for 100ml buffer)	y ml 0.2-Na2HPO4 (for 100ml buffer)	x ml 0.1M-citric acid (for 10ml buffer)	y ml 0.2-Na2HPO4 (for 10ml buffer)
4.79	3.2	57	43	5.7	4.3
4.97	3.4	56	44	5.6	4.4
4.98	3.6	55	45	5.5	4.5
5.06	3.8	54	46	5.4	4.6
5.19	4	53	47	5.3	4.7
5.24	4.2	52	48	5.2	4.8
5.45	4.4	51	49	5.1	4.9
5.49	4.6	50	50	5	5
5.55	4.8	49.3	50.7	4.93	5.07
5.6	5	48.5	51.5	4.85	5.15
5.8	5.2	46.4	53.6	4.64	5.36
6	5.4	44.25	55.75	4.425	5.575
6.1	5.6	42	58	4.2	5.8
6.3	5.8	39.55	60.45	3.955	6.045
6.57	6	36.85	63.15	3.685	6.315
6.68	6.2	33.9	66.1	3.39	6.61
6.78	6.4	30.75	69.25	3.075	6.925
6.98	6.6	27.25	72.75	2.725	7.275
7.13	6.8	22.75	77.25	2.275	7.725
7.43	7	17.65	82.35	1.765	8.235
7.53	7.2	13.05	86.95	1.305	8.695
7.7	7.4	9.15	90.85	0.915	9.085
7.9	7.6	6.35	93.65	0.635	9.365
7.93	7.8	5.65	94.35	0.565	9.435
8.04	8	4.95	95.05	0.495	9.505
8.03	8.2	4.25	95.75	0.425	9.575
8.26	8.4	3.55	96.45	0.355	9.645
8.26	8.6	2.85	97.15	0.285	9.715
8.33	8.8	2.15	97.85	0.215	9.785
8.36	9	1.45	98.55	0.145	9.855
8.63	9.2	0.75	99.25	0.075	9.925
8.7	9.4	0.05	99.95	0.005	9.995
8.76	stock bottle	0	100	0	10

References

• Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins Nature 394 192-195
• Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccaromyces cerevisiae reveals a relation between intracellular pH and growth Microbiology 155 268-278

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

• Megan N McClean 10:33, 5 June 2012 (EDT)Currently, we assay pHluorin with two separate filter cubes (with different excitation filters, but the same emission filter). Eventually we will move the excitation filters and the emission filter onto the microscope's filter wheels, so that alignment of the filter cubes in the turret is not an issue and the to cut down on the switching time.

Contact

• Megan N McClean 14:01, 05 June 12(EDT)

or instead, discuss this protocol.