Supplementary Methods
Additional materials
REAGENTS
BCA (bicinchoninic acid) protein assay kit (Pierce)
CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) (Sigma), 10% (w/v)
Chloramphenicol (Sigma), 34 mg/ml stock solution in ethanol
CYMAL-6 and CYMAL-7, 10% (w/v) (Anatrace),
Imidazole, for molecular biology, minimum 99% (Sigma)
Isopropyl -D-thiogalactopyranoside (IPTG) (Saveen), 1 M solution, filter sterilized
Kanamycin monosulfate (Sigma), 50 mg/ml stock solution, filter sterilized
Luria-Bertani Broth (LB medium) (Difco)
MgCl2, 1 M solution
N,N-dimethyldodecylamine N-oxide (lauryldimethylamine-oxide; LDAO) (Fluka), 10% (w/v)
n-decyl--D-maltopyranoside (DM) (Anatrace), 20% (w/v)
n-dodecyl--D-maltopyranoside (DDM) (Anatrace), 20% (w/v)
Ni-NTA Superflow (QIAGEN)
n-octyl--D-glucopyranoside (Anatrace), 20% (w/v)
n-undecyl--D-maltopyranoside (UDM) (Anatrace), 20% (w/v)
Pefabloc SC (Biomol)
Phosphate-buffered saline (PBS): 1.44 g Na2HPO4*2H2O (8.1 mM phosphate), 0.25 g KH2PO4 (1.9 mM
phosphate), 8.00 g NaCl, 0.2 g KCl in 1,000 ml H20; adjust pH to 7.4 using 1 M NaOH or 1 M HCl
Triton X-100 (Sigma), 20% (v/v)
Supplementary Protocol 1. Making CaCl2 competent cells and transformation.
1| Select the appropriate strain (see Supplemental table 2) and use 4-5 fresh colonies from a LB agar plate
to inoculate 100 ml of LB medium (in the case of pLysS, include the antibiotic chloramphenicol (34 µg/ml))
in a 250 ml conical flask.
2| Incubate the culture at 37°C, with shaking at 220 r.p.m.
3| When the culture has reached an OD600 of approx. 0.5 (after 2-3 h), transfer it to two 50 ml Falcon tubes.
4| Cool the culture on ice for 10 min.
5| Centrifuge the cells at 2,600g at 4°C for 10 min.
6| Decant the supernatant.
7| Resuspend the cell pellet gently in 10 ml of ice-cold 0.1 M CaCl2.
8| Incubate resuspended cells on ice for 10 min.
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9| Centrifuge the cells at 2,600g at 4°C for 10 min.
10| Decant the supernatant.
11| Resuspend the cell pellet gently in 2 ml of ice-cold 0.1 M CaCl2 plus 20% glycerol.
12| Incubate the resuspended cells on ice for 10 min.
13| Deliver 100 µl aliquots for future use (these competent cells can be used directly for a transformation:
go to Step 16).
14| Snap freeze aliquots in liquid nitrogen and store at –80 °C (competent cells can be stored at –80 °C for
at least 6 months).
15| Thaw frozen competent cells on ice for 5-10 min before mixing the cells with DNA.
16| To transform the expression vector containing the membrane protein GFP-fusion (see supplementary
Figure 1 and Supplementary Table 1) add 0.5 µl of expression vector (0.25 µg/µl of DNA) to 100 µl
competent cells
17| Incubate the mixture (that is, the cells with DNA) for 1 h on ice.
18| Heat shock cells for at 42 °C 1 min.
19| Place the cells for on ice two min.
20| Add 900 µl of LB medium to the cells and incubate at 37 °C for 1 h.
21| Centrifuge the cells at 2,600g for 1 min.
22| Remove 900 µl of the supernatant.
23| Resuspend the pelleted cells in the remaining 100 µl supernatant.
24| Plate the resuspended cells onto a LB agar plate with appropriate antibiotics (see supplementary Figure
1 and Table 1).
25| Incubate the plate at 37 °C overnight.
The plate can be stored at 4 °C.
Supplementary Protocol 2. Overexpression and isolation of GFP-8His
1| Overexpress GFP-8His in BL21(DE3)pLysS harboring pET20bGFP-8His for 4 hours at 25°C essentially
as described in Steps 21-26 of the main protocol. Add 0.4 mM IPTG (final concentration) for induction of
expression and ampicillin (100 µg/ml) rather than kanamycin.
2| Process cells as described in Steps 22-26 of the main protocol. Remove the supernatant, which contains
the GFP-8His, rather than the pellet for use in Step 3.
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3| Purify GFP-8His essentially as described in Steps 35-39 of the main protocol. Do not add detergent to
Buffers A and B, and wash the Ni-NTA column with 20 column volumes of 10 % Buffer B. Elute GFP-8His
with 50 % Buffer B.
4| Pool the fluorescent fractions (GFP-8His-containing) and dialyze overnight in 20 mM Tris-HCl, pH 7.5, 5
mM EDTA, pH 8, 100 mM NaCl.
GFP-His8 can be stored at –80°C in small aliquots. Avoid repeated freezing and thawing of GFP-8His
aliquots.
5| Determine protein concentration with the BCA assay and measure GFP fluorescence, as described in
Step 9 of the main protocol from 0.01 to 0.3 mg GFP-8His per ml.
Supplementary Protocol 3. Determining amount of membrane protein using GFP fluorescence
1| Plot the GFP fluorescence versus the protein concentration.
2| Use the slope of the plot determined in Step 1 to convert the GFP fluorescence from any 100 l sample
to mg of GFP-8His per ml.
3| To account for the dampening of GFP fluorescence in either whole-cells or non-detergent solubilized
membrane samples, multiply the values obtained in Step 2 by 1.5 for whole-cells and 1.3 for non-detergent
solubilized membranes (see Supplementary figure 2).
4| To estimate the amount of overexpressed membrane protein, divide the molecular weight of the protein
by 28 kDa (MW of GFP-8His) and multiply this by the amount of GFP-8His as determined in Steps 2-3.
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