Electrophoresis tech note 5705 Sensitivity and Protein-to-Protein Consistency of Flamingo™ Fluorescent Gel Stain Compared to Other Fluorescent Stains

Tom Berkelman, Life Science Group, Bio-Rad Laboratories, Inc., Hercules, CA 94547 UV transillumination. Differential protein-to-protein staining was Introduction examined using a collection of proteins selected to represent Proteomics research often involves 1-D or 2-D electrophoretic diversity in charge, size, and posttranslational modification. separation of protein mixtures in polyacrylamide gels. Methods Following electrophoresis, the gels are stained for detection Criterion™ Tris-HCl 4–20% gels with eighteen 5 mm wide and quantitation of the separated proteins. Fluorescent wells were used for SDS-PAGE. Gel images generated by stains are commonly used for this purpose due to the high laser scanning were produced using the Molecular Imager® sensitivity and wide dynamic range possible using fluorescent PharosFX™ system. Gels were scanned using either 532 nm reagents. Quantitation is most reliable when the fluorescence or 488 nm excitation as indicated. Fluorescence emission response is linear with respect to the amount of protein over on all scans was filtered through a 605 nm bandpass filter. the widest possible range. Ideally, a fluorescent stain should Except when indicated, the photomultiplier tube (PMT) setting also respond similarly to diverse proteins. There should be for low-intensity samples was used. Gel images generated minimal specificity with respect to the physical or chemical by UV transillumination were produced using the VersaDoc™ characteristics of an individual protein. 3000 imaging system with 300 nm bulbs, 520 nm longpass emission, and the camera aperture fully open. All images A number of fluorescent protein stains are now available. The were analyzed using Quantity One® software. current study was undertaken to compare the performance of four commercial staining products: Flamingo fluorescent gel Experiments to determine relative signal and background stain (Bio-Rad Laboratories), SYPRO Ruby protein gel stain levels, visual limits of sensitivity, and linearity used gels loaded (Invitrogen Corp., also distributed by Bio-Rad Laboratories), with a dilution series of broad range SDS-PAGE standards Deep Purple total protein stain (GE Healthcare), and Krypton (unstained). Dilutions were prepared to give the following protein stain (Pierce Biotechnology). sample load (for each protein) in a loading volume of 5 µl: 960 ng, 480 ng, 240 ng, 120 ng, 60 ng, 30 ng, 15 ng, 8 ng, Detection sensitivity is related to the fluorescence intensity 4 ng, 2 ng, 1 ng, 0.5 ng, 0.25 ng, 125 pg, 60 pg, 30 pg. of a given quantity of stained protein. It also depends on the magnitude of background fluorescence. These values were The proteins selected to evaluate protein-to-protein variability determined for the different stains under identical imaging in staining intensity were resolved as single bands by SDS- conditions. The limit of sensitivity and the linearity of response PAGE. Constant 240 ng loads based on A280 in a guanidine for each stain were determined using visible-light laser solution (Gill and von Hippel 1989) were applied to SDS-PAGE imaging under conditions optimal for each stain. The limits of and stained as described. All of the proteins were purchased sensitivity were also determined for an imaging system using from Sigma. Gels were stained according to protocols accompanying each product as summarized in Table 1.

Table 1. Summary of gel staining protocols. Flamingo Fluorescent Gel Stain SYPRO Ruby Deep Purple Krypton Protein Stain 1. Fix 2 hr in 40% ethanol, 10% 1. Fix 2x 30 min in 50% methanol, 1. Fix 1 hr in 10% methanol, 1. Fix 2x 30 min in 40% ethanol, acetic acid 7% acetic acid 7% acetic acid 10% acetic acid 2. Stain overnight (15–18 hr) in stain 2. Stain overnight (15–18 hr) in stain 2. Wash 30 min in 200 mM 2. Wash 5 min in water solution solution sodium carbonate 3. Stain 2 hr in stain solution 3. Wash 30 min in 0.1% (v/v) 3. Destain 30 min in 10% methanol, 3. Stain 1 hr in stain solution 4. Destain 5 min in 5% acetic acid Tween 20 7% acetic acid 4. Destain 2x 15 min in 5. Wash 2x 15 min in water 4. Wash 2x 5 min in water 7.5% acetic acid Results Fluorescence Intensity and Background Flamingo fluorescent gel stain is excited in the visible range with an excitation peak intermediate between the 532 nm and 488 nm lasers used by the Pharos FX imager. Therefore, both excitation wavelengths were tested on gels stained with Flamingo stain as well as on gels stained with SYPRO Ruby stain. Gels stained with Flamingo fluorescent gel stain had the highest protein-associated fluorescence intensity. Gels stained with Krypton and Deep Purple stains had considerably lower background than those stained with either Flamingo or SYPRO Ruby stains but had correspondingly lower protein-associated fluorescence. Flamingo stain gave the highest difference between signal and background.

Flamingo — scanned with 532 nm laser SYPRO Ruby — scanned with 532 nm laser Deep Purple — scanned with 532 nm laser ng ng ng 960 480 240 120 60 30 15 8 4 2 1 0.5 0.25 960 480 240 120 60 30 15 8 4 2 1 0.5 0.25 960 480 240 120 60 30 15 8 4 2 1 0.5 0.25

Flamingo — scanned with 488 nm laser SYPRO Ruby — scanned with 488 nm laser Krypton — scanned with 532 nm laser ng ng ng 960 480 240 120 60 30 15 8 4 2 1 0.5 0.25 960 480 240 120 60 30 15 8 4 2 1 0.5 960 480 240 120 60 30 15 8 4 2 1 0.5 0.25 0.25

Fig. 1. Images of gels stained with different fluorescent stains generated with the PharosFX system. Gels stained with Flamingo and SYPRO Ruby stains were imaged under both 488 nm (blue) and 532 nm (green) laser light. Gels stained with Deep Purple and Krypton stains were imaged only under 532 nm (green) laser light. All images were subjected to the same image transformation.

Table 2. Protein-associated and background fluorescence. Fluorescence intensity values are given in arbitrary relative units. Protein-associated fluorescence equals pixel intensity of protein lane minus background pixel intensity. Pixel Intensity Protein– Gel Stain Excitation Protein Associated Wavelength Lane Background Fluorescence Flamingo (532 nm) 49.74 4.36 45.92 Flamingo (488 nm) 43.63 3.76 39.87 SYPRO Ruby (532 nm) 22.56 4.44 18.12 SYPRO Ruby (488 nm) 43.03 7.67 35.36 Fig. 2. Determination of fluorescent signal associated with protein and Deep Purple (532 nm) 24.91 1.01 23.89 background fluorescence (example). A box encompassing all of the standards loaded at 240 ng (U1), and a box of equal area around an empty Krypton (532 nm) 26.26 0.72 25.54 region (U2) were drawn on each gel. The total pixel intensity in each region was determined. Results are shown in Table 2. The example gel shown was stained with Flamingo fluorescent gel stain.

© 2008 Bio-Rad Laboratories, Inc. Bulletin 5705 Sensitivity With Visible Light Laser Scanning Table 3. Limit of detection with visible laser light excitation for each Flamingo fluorescent gel stain gave the lowest limit of staining method. The value given is the lowest amount of protein for which all nine of the protein standards are visible. detection with laser scanning. Gel Stain Flamingo SYPRO Ruby Deep Purple Krypton Wavelength 532 nm 488 nm 532 nm 532 nm Protein amount 30 pg 500 pg 125 pg 125 pg

FlamingoFlamingo (532(532 nm) nm) SYPROSYPRO Ruby Ruby (488 (488 nm) nm) DeepDeep Purple Purple (532(532 nm) nm) KryptonKrypton (532 (532 nm) nm)

60 pg 30 pg 60 pg 30 pg 1 ng 0.50.5 ngng0.250.25 ngng125125 pg pg6060 pg pg30 30pg pg 1 ng 0.5 ngng0.250.25 ng ng125 pgpg60 pgpg 3030 pg 1 ngng 0.50.5 ng0.250.25 ng ng125125 pg 60pg pg30 pg 11 ng 0.50.5 ng ng0.250.25 ng 125ng125 pg60 pg pg30 pg Fig. 3. Gel images generated by laser scanning. Gels contain- ing a dilution series of protein standards were stained either with Flamingo, SYPRO Ruby, MyosinMyosin Deep Purple, or Krypton stains as ß-Galactosidaseß-Galactosidase described and scanned PhosphorylasePhosphorylase b b using the PharosFX system. BSA BSA The images were transformed to OvalbuminOvalbumin show the lowest possible visual limit of sensitivity. Only the portion CACA of each gel showing protein loads TrypsinTrypsin inhibitor inhibitor of 1 ng or lower is shown. Table 3 LysozymeLysozyme AprotininAprotinin summarizes the results.

Linearity of Staining Flamingo fluorescent gel stain exhibited the best linearity over the widest range of quantities, with 2R >0.99 over the entire range tested (Figure 5).

Flamingo SYPROSYPRO Ruby Ruby Deep Purple Krypton FlamingoFlamingoFlamingo (532 nm) SYPRO Ruby (488 nm) DeepDeep Purpl Purplee KryptoKryptonn 700,000 1,000,000 700,000 700,000700,000700,000 1,000,0001,000,001,000,0000 1,000,001,000,0000 700,000700,000 600,000 600,000 2 22 2 2 e 600,000600,000600,000 R = 0.996 800,000 R22 = = 0.98 0.9855 800,000 R22 = 0.993 600,000600,000 R22 = 0.9809 e e 800,000800,000800,000 RR = = 0.98 0.9855 800,000800,000 RR = = 0.993 0.993 500,000 RR = = 0.9809 0.9809 500,000500,000 500,000500,000 HighHigh range range 500,000 600,000 600,000 400,000 HighHigh rangrangee 400,000400,000 600,000600,000600,000 600,000600,000 400,000400,000 (30–960 ng) 300,000300,000 400,000 300,000 300,000300,000 400,000400,000400,000 400,000400,000 300,000300,000 (30–960 ng) 200,000200,000 200,000 (30–960(30–960 ngng)) Fluorescenc 200,000200,000 200,000 200,000200,000 Fluorescenc Fluorescenc 200,000 100,000 200,000200,000200,000 200,000200,000 100,000 Fluorescence 100,000100,000100,000 100,000100,000 0 00 00 00 00 00 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 00 200200 400400 600600600 800800 100010001000 120012012000 00 200200 400400 600600 800800 10001000 12001200 00 2 20000 404000 6 60000 808000 1 1000000 12001200 00 2 20000 400400 606000 8 80000 10001000 12001200

60,000 140,000 80,000 120,000 60,00060,0060,0000 140,000140,000140,000 80,00080,000 120,00120,0000 70,000 50,000 22 120,000 22 70,00070,000 2 100,000 2 e 50,00050,0050,0000 RR2 2== 0.9990.999 120,000120,000120,000 R22 = = 0.97 0.9799 R22 = 0.988 100,000100,000 R22 = 0.9813 e e RR = = 0.999 0.999 RR = = 0.97 0.9799 60,000 RR = = 0.98 0.9888 RR = = 0.9813 0.9813 40,000 100,000100,000100,000 60,00060,000 80,000 MiMidd range range 40,00040,0040,0000 80,000 50,00050,000 80,00080,000 MiMidd rangerange 30,000 80,00080,0080,0000 40,000 60,000 (2–60 ng) 30,00030,0030,0000 60,000 40,00040,000 60,00060,000 20,000 60,00060,0060,0000 30,000 (2–60 ng) 20,0020,0000 40,000 30,00030,000 40,00040,000 (2–60(2–60 ngng)) Fluorescenc 20,000 40,00040,0040,0000 20,000

Fluorescenc Fluorescenc 20,00020,000 10,0010,0000 20,000 10,000 20,000 Fluorescence 10,000 20,00020,0020,0000 10,00010,000 20,00020,000 0 0 00 00 00 00 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 00 1 11000 2 20020 3 30030 4 40050 5 50060 6 6 0070 7 700 00 1 10010 2 20020 3 30030 4 40050 5 50 060 6 60 0 7 77000 00 1 100 2 200 3 300 4 400 5 500 6 600 7 700 00 1 100 2 200 3 300 4 400 5 500 6 600 7 700

4,000 4,0004,004,0000 30,00030,0030,0000 10,00010,000 16,0016,0000 3,500 14,000 3,503,5000 22 25,000 22 2 2

e 3,500 R = 0.994 14,0014,0000 R22 = 0.994 25,00025,0025,0000 R22 = = 0.91 0.9133 8,000 R22 = 0.975 R22 = 0.9934 e e 3,000 RR = = 0.994 0.994 3,0003,003,0000 RR = = 0.91 0.9133 8,008,0000 RR = = 0.97 0.9755 12,0012,0000 RR = = 0.993 0.99344 Low range 2,500 20,000 Low range 2,5002,502,5000 20,00020,0020,0000 6,000 10,0010,0000 LoLoww rangerange 2,000 6,006,0000 (0.125–4 ng) 2,0002,002,0000 15,00015,0015,0000 8,008,0000 1,500 4,004,0000 6,000 (0.125–4 ng) 1,5001,501,5000 10,0010,0000 6,006,0000 (0.125–4(0.125–4 ngng)) Fluorescenc 1,000 10,000 4,000

Fluorescenc Fluorescenc 1,0001,001,0000 4,004,0000 500 5,000 2,002,0000 Fluorescence 500500 5,0005,005,0000 2,000 5000 2,002,0000 00 00 00 00 0 1 2 3 4 5 00 1 2 3 4 5 0 1 2 3 4 5 00 1 1 2 2 33 4 4 55 00 1 11 222 333 4 4 55 5 00 1 1 22 3 3 44 5 5 00 1 1 2 2 33 4 4 5 5 ng ovalbumin ngng ovalbumi ovalbuminovalbuminn ngng ovalbumin ovalbumin ngng ovalbumi ovalbuminn 500500500 2 R 2 = 0.993

e 400400 R22 = 0.993 Very low range e e 400400 RR = = 0.993 0.993 Very low range 300300 Ve(30–250Veryry lowlow pg) rangrangee 300300 NA (30–250 pg) 200200200

(30–250(30–250 pgpg)) Fluorescenc

Fluorescenc Fluorescenc 100 NA NA NA 100100100 NANA NANA NANA Fluorescence 00 00 0 0.050.05.05 0.0.110.1 0 0.15.15 0.150. 0.220.25 0 0.2.25 5 0.3 0 0.3.3 ngngng ovalbuminovalbumi ovalbuminn

© 2008 Bio-Rad Laboratories, Inc. Bulletin 5705 Limit of Sensitivity With UV Transillumination Table 4. Limit of sensitivity with uv transillumination for each staining SYPRO Ruby fluorescence was considerably brighter than method. The value given is the lowest amount of protein at which all nine of the protein standards are visible. the other stains under UV transillumination and needed a Gel Stain shorter exposure to prevent image saturation. Flamingo stain Flamingo SYPRO Ruby Deep Purple Krypton gave the lowest limit of sensitivity with UV transillumination. Protein amount 125 pg 500 pg 500 pg 250 pg

FlamingoFlamingo (180 sec) sec) SYPROSYPRO RubyRuby (20 (20 sec) sec) DeepDeep Purple (180 (180 sec) sec) KryptonKrypton (180 (180 sec) sec)

4 ng4 ng2 2ng ng 11 ng ng 0.5 ngng0.250.25 ng125125 pg pg 4 ng 22 ngng 11 ngng 0.5 ng0.250.25 ng125125 pg pg 44 ngng 2 ng 11 ng ng 0.50.5 ng ng0.250.25 ng125 ng125 pg pg 44 ng ng 2 2ng ng 1 1ng ng0.50.5 ng 0.25ng 0.25 ng125 ng 125pg pg Fig. 4. Gel images generated by UV transillumination. Gels containing dilution series of protein standards were stained either with Flamingo, MyosinMyosin SYPRO Ruby, Deep Purple, or Kryp-

ß-Galactosidaseß-Galactosidase ton stains as described and imaged PhosphorylasePhosphorylase b b using the VersaDoc 3000 system BSABSA with exposure times as indicated. The OvalbuminOvalbumin images were transformed to show the lowest possible visual limit of CA CA sensitivity. Only the portion of each TrypsinTrypsin inhibitor inhibitor gel showing protein loads of 4 ng or LysozymeLysozyme AprotininAprotinin lower is shown. Table 4 summarizes the results.

Fig. 5. Linear dynamic range of Flamingo SYPROSYPRO Ruby Ruby SYPRO Ruby FlamingoFlamingo DeepDeep DeepPurpleFlamingo Purpl Purpl (532ee nm) KryptonKryptoKrypto (532nn nm) staining intensity. GelsDeep containing Purpl e Krypton 700,000700,000 1,000,001,000,0000 1,000,0001,000,001,000,00700,00000 1,000,00700,000700,000700,0000 1,000,00dilution0 series of protein standards 700,000 were stained either with Flamingo, 600,000600,000 222 600,000 R22 22= 0.993 600,000600,000600,000 R22 = 0.9809 2 600,000 2 e

e e e e 800,000 e 800,000800,000 RR = == 0.98 0.980.9855 800,000800,000800,000 RR = == 0.993 0.9930.993 800,000 RR = == 0.980.9809 0.98090.98095 800,000 R = 0.993 R = 0.9809 500,000500,000 500,000 500,000500,000500,000 SYPRO Ruby, Deep Purple, or 500,000 HighHigh rangrangee 400,000400,000 600,000600,000 High range 600,000400,000600,000600,000 400,000400,000600,000400,000 600,000Krypton stains as described and 400,000 300,000 300,000300,000 400,000400,000 400,000300,000400,000400,000 300,000400,000300,000 400,000scanned at 532 nm or 488 nm using 300,000 (30–960(30–960 ngng)) 200,000200,000 (30–960 ng) 200,000 200,000200,000200,000 200,000 Fluorescenc Fluorescenc Fluorescenc Fluorescenc Fluorescenc Fluorescenc 200,000200,000 200,000200,000 200,000 200,000the PharosFX system. The Flamingo- 100,000 200,000 100,000100,000100,000 100,000 100,000100,000 100,000 stained gel was scanned using the 00 00 00 000 0 0 00 200200 400400 600600 800800 10001000 12012000 00 200200 400400 600600 800800 10001000 12001200 00 200 2 20000 400404000 600600 6 60000 800808000 10001000 1 1000000 1200120120012000 00 200 2200 20000 400400 600606006000 800 8800 80000 10001000 120012001200 instrument0 setting 200 for40 medium-0 600 800 1000 1200 0 200 400 600 800 1000 1200 intensity samples (45% maximum PMT voltage). All other gels were 60,0060,0000 140,000140,000 80,00060,0080,00080,0000 120,000120,00140,000120,0000 80,000 120,000 70,00070,00070,000 2 2 70,000scanned using the instrument 50,0050,0000 222 120,000120,000 222 50,0070,0000 R 2 22= 0.988 100,000120,000100,000 R222 = 0.9813 2 100,000 2

e 100,000 e e e e e RR = == 0.999 0.9990.999 RR = == 0.97 0.970.9799 RR = == 0.9990.98 0.980.9888 RR = == 0.970.9813 0.98130.98139 R = 0.988 R = 0.9813 100,000100,000 60,00060,00060,000 100,000 60,000setting for low-intensity samples 40,0040,0000 50,00040,0050,00050,0000 80,00080,00080,000 50,000 80,000 MiMidd rangerange 80,0080,0000 Mid range 50,000 80,000 (55% maximum PMT voltage). 30,0030,0000 40,00030,0040,00040,0000 60,00060,00060,000 40,000 60,000 60,0060,0000 60,000 The total pixel intensity following 20,0020,0000 30,00020,0030,00030,0000 40,000 30,000 (2–60(2–60 ngng)) 20,000 40,0040,0000 (2–60 ng) 40,00040,00040,0040,0000 40,000

Fluorescenc background subtraction associated Fluorescenc Fluorescenc Fluorescenc Fluorescenc Fluorescenc 20,00020,00020,000 20,000 20,000 10,0010,0000 20,0020,0000 10,00010,0010,00010,0000 20,00020,0020,0000 10,000with the ovalbumin band was 20,000 0 00 00 00 00 determined0 across the entire 0 00 1 100 2 200 3 300 4 400 5 500 6 600 7 700 00 1 100 2 200 3 300 4 400 5 500 6 600 7 700 00 1 11000 2 20020 3 30030 4 40050 5 50060 6 6 0070 7 700 00 1 1 100100 2 2 200020 3 3 300030 4 4 400050 5 550 0 060 6 6 60 0 0 7 7700 700 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 visible range of the dilution series. Fluorescence is given in arbitrary 4,004,0000 30,0030,0000 10,00010,00010,0004,000 16,00016,0030,0016,00000 10,000relative units. Results are graphed 16,000 3,503,5000 3,500 2 14,000 2 222 25,0025,0000 222 R 2 22= 0.975 14,00014,0025,0014,00000 R222 = 0.9934 2 14,000 2 e e e e e e RR = == 0.994 0.9940.994 8,000 R = 0.994 3,003,0000 RR = == 0.91 0.910.9133 8,0003,008,008,0000 RR = == 0.97 0.970.9755 12,00012,0012,0000 RR = == 0.910.993 0.9930.993344 over8,000 four concentrationR = 0.975 ranges. 12,000 R = 0.9934 20,0020,0000 20,000 LoLoww rangerange 2,502,5000 Low range 6,0002,506,006,0000 10,00010,0010,0000 6,000 10,000 2,002,0000 15,0015,0000 2,000 15,008,0008,008,00000 8,000 1,501,5000 4,5001,504,004,0000 4,000 (0.125–4(0.125–4 ngng)) 1,500 10,0010,0000 (0.125–4 ng) 10,006,0006,006,00000 6,000 Fluorescenc Fluorescenc Fluorescenc Fluorescenc Fluorescenc Fluorescenc 1,001,0000 1,000 4,0004,004,0000 4,000 2,0002,002,0000 2,000 500500 5,005,0000 500 2,0002,005,002,0000 2,000 00 00 00 00 0 0 00 1 1 2 2 33 4 4 55 00 1 1 22 33 4 4 5 5 00 1 1 2 2 3 3 4 4 5 5 00 1 1 1 2 2 2 333 4 4 4 5 5 5 0 1 2 3 4 5 0 1 2 3 4 5 ngng ovalbumi ovalbuminn ngngng ovalbumin ovalbumin ngngng ovalbumiovalbumi ovalbuminovalbuminnn ng ovalbumin ng ovalbumin 500500 500

222 2 e e e e e e 400400 RR = == 0.993 0.9930.993 400 R = 0.993 VeVeryry lowlow rangrangee 300300 Very low range 300 NA NA (30–250(30–250 pgpg)) 200200 (30–250 pg) 200 Fluorescenc Fluorescenc Fluorescenc Fluorescenc Fluorescenc Fluorescenc 100100 NANA 100 NANA NANA NA NA 00 0 00 0 0.05.05.05 0.0.11 0 0.15.15.15 0.0.22 0 0.2.2.255 0 0.3.3.3 0 0.05 0.1 0.15 0.2 0.25 0.3 ngng ovalbumi ovalbuminn ng ovalbumin

© 2008 Bio-Rad Laboratories, Inc. Bulletin 5705 Protein-to-Protein Differential Staining 2.52.5 All of the stains tested exhibited significantly reduced staining Flamingo Deep Purple 2.02.0 SYPRO Ruby Krypton intensity with the very acidic protein pepsin (Figure 6). Otherwise, Flamingo, SYPRO Ruby, and Krypton stains did 1.51.5 not exhibit any readily discernible staining bias based on size 1.01.0 or isoelectric point (pI). None of the stains tested appeared to

Normalized fluorescence 0.5 exhibit any bias related to posttranslational modification. All 0.5 Normalized fluorescence reacted similarly with RNase A and RNase B, which only differ 0 by the presence of glycosylation on RNase B. Deep Purple PepsinPepsin AprotininAprotinin RNaseRNase BBRNaseRNase A A UbiquitinUbiquitinAmylaseAmylase LysozymeLysozymeOvalbumin stain exhibited reduced staining intensity with the acidic protein Ovalbumin CalmodulinCalmodulin Apotransferrin SerumSerum albumin albumin Apotransferrin Acid-Acid glycoprotein glycoprotein calmodulin and almost no staining with the very acidic protein -a 1 Myelin basic protein a 1 Myelin basic protein pepsin. Flamingo, SYPRO Ruby, and Krypton stains exhibited Fig. 6. Consistency of staining response among diverse proteins. similar average deviations from mean staining intensity, with A group of proteins was selected to represent diverse properties Flamingo stain showing the lowest average deviation. Deep (described in Table 5). A constant amount (240 ng) of each protein was Purple exhibited a significantly greater average deviation from resolved by SDS-PAGE. Gels were stained either with Flamingo, SYPRO Ruby, Deep Purple, or Krypton stains and scanned with the PharosFX system mean staining intensity than did the others. using the 488 nm laser for SYPRO Ruby and the 532 nm laser for the other stains. Total fluorescence associated with each protein band was determined, and the value was normalized by dividing by the average total fluorescence associated with each protein band for each gel.

Table 5. Proteins selected for differential staining experiment. Table 6. Average deviation from mean staining intensity within the Protein Special Properties group of proteins tested. Aprotinin (bovine) Small (MW = 6,500) Gel Stain Basic (pI = 9.24) Flamingo SYPRO Ruby Deep Purple Krypton Protein amount 23.5% 25.2% 35.5% 25.0% Lysozyme (chicken) Basic (pI = 9.32) Ovalbumin (chicken) Phosphoprotein Glycoprotein RNase B (bovine) Basic (pI = 8.64) Glycoprotein RNase A (bovine) Basic (pI = 8.64) a1-Acid glycoprotein (ovine) Acidic (pI ~ 4) Glycoprotein Myelin basic protein (bovine) Very basic (pI = 11.28) Phosphoprotein Serum albumin (bovine) Pepsin (porcine) Very acidic (pI = 3.24) Phosphoprotein Ubiquitin (bovine) Small (MW = 8,600) Amylase (Bacillus amyloliquifaciens) Apotransferrin (human) Glycoprotein Calmodulin (bovine) Acidic (pI = 4.09) Discussion stain was notable in its almost complete inability to stain Of the stains tested, Flamingo fluorescent gel stain had pepsin and its relative lack of response to calmodulin, another the brightest intrinsic fluorescence when scanned with the very acidic protein. This may be explained by the reported PharosFX laser-based imager regardless of whether 488 nm requirement for interaction between the dye in Deep Purple or 532 nm excitation was used. A lower scanner PMT setting and the primary amine of lysine residues (Coghlan et al. was therefore necessary in order to prevent image saturation 2005). The other acidic protein tested, a1-acid glycoprotein, when evaluating the linear range of Flamingo fluorescent gel stained quite strongly with Deep Purple. This acidic stain. When this measure was taken, Flamingo fluorescent glycoprotein owes its low pI to the presence of sialic acid gel stain exhibited a significantly wider linear range for rather than a lack of basic amino acids, suggesting that the quantitation, and better linearity across the entire range than mechanistic basis of Deep Purple staining is more related to did the other stains tested. amino acid composition than overall charge.

SYPRO Ruby protein gel stain was notable in that its optimal Conclusions exposure time for imaging by UV transillumination was Of the stains tested: considerably shorter than that required for imaging the n Flamingo fluorescent gel stain has the highest fluorescence other stains (20 sec vs. 180 sec). This is consistent with with visible light laser scanning and gives the highest signal the reported high relative UV absorbance of SYPRO Ruby to background ratio (Berggren et al. 2000). Nonetheless, SYPRO Ruby was no more sensitive with UV transillumination than were the other n Flamingo stain is the most sensitive for use with visible light tested stains when their relative lack of brightness was laser scanning compensated for with a longer exposure. Both Flamingo n Flamingo stain is the most sensitive for use with UV fluorescent gel stain and Krypton protein stain proved more transillumination, provided that a relatively long exposure sensitive with UV transillumination, with Flamingo stain time is used showing the highest sensitivity. This may be due to the n Flamingo stain exhibits the best signal vs quantity linearity relatively high background observed with SYPRO Ruby. n Flamingo, SYPRO Ruby, and Krypton stains all exhibit However, SYPRO Ruby may be preferable in instances where relatively low protein-to-protein staining variability, with equipment limitations prevent the relatively long exposure Flamingo stain showing the least. Deep Purple total protein times required by the other stains. stain exhibits more variability Factors considered as possibly influencing staining behavior If the primary concerns in choosing a stain are sensitivity, were charge (pI) and the presence of posttranslational linearity, and protein-to-protein consistency, Flamingo stain is modifications (glycosylation and phosphorylation). Two small the preferred one. proteins (aprotinin and ubiquitin) were also included in the study to test for any possible bias towards or against small References Berggren K et al., Background-free, high sensitivity staining of proteins in proteins. The only bias observed for all stains was poor one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a staining of pepsin. Pepsin is exceptional among proteins luminescent ruthenium complex, Electrophoresis 21, 2509–2521 (2000) for its very low pI and its low content of basic amino acids. Coghlan DR et al., Mechanism of reversible fluorescent staining of protein with It therefore displays little positive charge, suggesting that epicocconone, Org Lett 7, 2401–2404 (2005) electrostatic interaction between negatively charged dye and Gill SC and von Hippel PH, Calculation of protein extinction coefficients from amino acid sequence data, Anal Biochem 182, 319–326 (1989) positive charges on proteins may be a staining mechanism shared among the stains tested. Deep Purple total protein SYPRO is a trademark of Molecular Probes, Inc. Deep Purple is a trademark of GE Healthcare. Krypton is a trademark of Pierce Biotechnology, Inc. Tween is a trademark of ICI Americas Inc.

Information in this tech note was current as of the date of writing (2006) and not necessarily the date this version (rev A, 2008) was published.

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