WesternBright Quantum

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Jocelin Murphy
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1 WesternBright Quntum Quntify hemiluminesent Western lots over wie ynmi rnge WesternBright Quntum is new hemiluminesent regent speilly formulte for CCD imging. This novel Horserish peroxise (HRP) sustrte provies strong, long-lsting signl, the roest useful liner rnge n high sensitivity for the most quntittive hemiluminesent Western ssys. Chemiluminesene is the metho of hoie for sensitive Western lot etetion, ut hs not een onsiere quntittive, primrily euse of the limite liner ynmi rnge of film, n of ommerilly ville sustrtes. WesternBright Quntum HRP sustrte overomes the limittions of other sustrtes, showing no sustrte epletion t high protein los. WesternBright Quntum is speilly formulte for quntittive hemiluminesent Western lotting, prouing liner signl over ro rnge of protein onentrtions spnning 3 orers of mgnitue. Comine with CCD imging, whih provies muh greter liner ynmi rnge thn film, WesternBright Quntum llows highly quntittive t to e otine from hemiluminesent Western lots. The roest liner rnge for the most powerful quntittion Aurte omprison of the intensities of ifferent protein ns requires tht the ns e within the liner ynmi rnge of etetion, whih is the rnge of onentrtions from the fintest n tht n e etete to the most intense n for whih the signl is not sturte. When use to etet Western lot ontining seril ilution of trnsferrin protein Figure 1. The priniple of hemiluminesent Western lotting. A seonry ntioy, onjugte to horserish peroxise (HRP) ins to primry ntioy irete towrs the protein of interest. The lot is inute with hemiluminesent sustrte, whih is onverte y HRP into light emitting luminesent moleule ng 4.66 ng 2.33 ng 1.16 ng 0.58 ng 0.29 ng 0.14 ng 72.9 pg 36.4 pg 18.2 pg 9.1 pg 4.6 pg 2.3 pg 1.1 pg 0.6 pg 0.3 pg e Figure 2. Liner ynmi rnge of WesternBright Quntum. Ientil Western lots ontining seril ilutions of trnsferrin were proe with rit-nti-trnsferrin primry ntioy, n got-nti-rit seonry ntioy onjugte to Horserish peroxise. The lots were inute with hemiluminesent sustrtes s reommene y eh mnufturer. All lots were simultneously imge for 2 minutes on CCD imger n ll isply prmeters re ientil ross ll imges shown in the figure. WesternBright Quntum shows the lrgest ynmi rnge out of ll four sustrtes with the highest R 2 vlue.. Amershm ECL (GE Helthre);. Amershm ECL Plus (GE Helthre);. SuperSignl West Dur (Thermo Sientifi);. WesternBrights Quntum; e. Signl intensity vs protein mount plot showing est fit liner regression for ll four sustrtes. Bns tht fll on the liner prt of the urve re inite. Pge 1 of 5

2 (Figure 2), WesternBright Quntum provies the roest liner ynmi rnge when ompre to severl other ommerilly ville hemiluminesent sustrtes (Figure 2e, Tle 1). Notly, no sustrte epletion is seen t ny protein los with WesternBright Quntum, while sustrte epletion interferes with etetion of high mounts of protein y oth ECL Plus n SuperSignl West Dur (seen s reverse intensity ns in Figures 2 n 2). The ility to etet high mounts of protein without sustrte epletion ontriutes to the inrese ynmi rnge provie y WesternBright Quntum. For etermining liner rnge, est fit t nlysis ws performe using liner regression. Dt points orresponing to high protein mounts were exlue from tsets one y one s outliers to otin the roest rnge with R 2 vlue ove Using this metho, WesternBright Quntum proue the roest liner rnge with the highest R 2 vlue. Sustrte WesternBright Quntum SuperSignl West Dur ECL Plus ECL Liner Dynmi Rnge 4.6 pg 4.7 ng 9.1 pg 2.3 ng 9.1 pg 0.29 ng 73 pg 1.2 ng Tle 1. Liner ynmi rnge for four hemiluminesent sustrtes. The liner ynmi rnge inlues t points tht proue the est possile liner regression fit (Figure 2e). Anlysis of the experiment epite in Figure 2. Long lsting signl CCD exposure times with WesternBright Quntum re s quik s film exposures with other sustrtes. In ition, WesternBright Quntum provies greter signl stility thn the ompetition, llowing long-term CCD mer exposures to e onute, if esire, to etet fint ns n low-unne proteins. The long-lsting signl lso mens there is no nee to rush to imge lot, sine the signl will remin strong for severl hours. To follow signl strength over time, GAPDH ws etete on quruplite Western lots ontining seril ilutions of HeL ell lyste (Figure 3). A n whih ws etermine to e within the liner rnge of ll four hemiluminesent sustrtes teste ws quntifie t severl time points over the next 10 hours. WesternBright Quntum mintine signl strength, with signl elining only pproximtely 30% over 60 minute spn, while the signl from ECL, ECL Plus n SuperSignl West Dur eh eye y lmost 90%, to rely etetle levels, over the sme perio (Figure 3). In ft, the WesternBright Quntum signl is still etetle in 2 minute exposure 10 hours lter (inset, Figure 3 n 3). 5 min 60 min 10 hr Figure 3. Signl urtion of WesternBright Quntum. Blots etete using WesternBright Quntum n three other hemiluminesent sustrtes were imge simultneously t severl times over 10 hour perio. Eh exposure ws 2 min long for ll lots n ll sustrtes. Intensities of ns ontining the sme mount of protein re plotte for eh sustrte in 3. Imges of this n on the WesternBright Quntum lot otine fter 5 min (3), 60 min (3) n 10 hr (3) re shown. Pge 2 of 5

Low kgroun for high sensitivity WesternBright Quntum proues n exeptionlly strong signl with little to no kgroun, for high signl to noise rtio n exellent sensitivity.

To emonstrte the reltive lk of kgroun with WesternBright Quntum ompre to other hemiluminesent sustrtes, quruplite Western lots were etete using vrious HRP sustrtes s esrie in Methos.

After 20 minute exposure, the lot etete using WesternBright Quntum remins ler of kgroun (Figure 4) ompre to ECL Plus (Figure 4) or SuperSignl West Dur (Figure 4).

3 Low kgroun for high sensitivity WesternBright Quntum proues n exeptionlly strong signl with little to no kgroun, for high signl to noise rtio n exellent sensitivity. The low kgroun is espeilly pprent when WesternBright Quntum is is use with film etetion. To emonstrte the reltive lk of kgroun with WesternBright Quntum ompre to other hemiluminesent sustrtes, quruplite Western lots were etete using vrious HRP sustrtes s esrie in Methos. The four lots were simultneously expose to single film. After 20 minute exposure, the lot etete using WesternBright Quntum remins ler of kgroun (Figure 4) ompre to ECL Plus (Figure 4) or SuperSignl West Dur (Figure 4). Figure 5 emonstrtes the superior sensitivity of WesternBright Quntum on film; uplite lots were etete using WesternBright Quntum or SuperSignl West Pio. In short exposure, WesternBright Quntum (Figure 5) etets n of trunte protein not visile with the other regent (Figure 5) unless longer exposure is use (Figure 5). Aurtely quntify low n high unne proteins WesternBright Quntum's high sensitivity, low kgroun n ro liner rnge omine to llow the urte quntittion of low n high unne proteins in single experiment. Low unne proteins n e etete ue to the exellent sensitivity, n the ro liner ynmi rnge llows high unne proteins to e etete with the sme exposure, without sturtion of signl. Figure 6 emonstrtes the superior performne of WesternBright Quntum when proing for either high unne (tin) or low unne (STAT-1) protein. Seril ilutions of extrts from A431 ells were ssye y replite Western lots, proe for tin n STAT- 1, n visulize using WesternBright Quntum or other ommerilly ville hemiluminesent sustrtes s esrie in Methos. For quntittive etetion on the liner prt of the intensity vs protein urve, WesternBright Quntum prove to e 8-times more Figure 4. Extremely low kgroun with WesternBright Quntum. Duplite Western lots were evelope using () ECL, () ECL Plus, () SuperSignl West Dur, or () WesternBright Quntum s sustrtes. After 20 minute exposure to film, WesternBright Quntum isplys the est omintion of sensitivity n signl with low kgroun. All isply prmeters re ientil ross ll imges shown in this figure. WesternBright Quntum 45 se exposure SuperSignl West Pio 45 se exposure Figure 5. Superior sensitivity of WesternBright Quntum with film etetion. WesternBright Quntum () or SuperSignl West Pio (Thermo Sientifi) (, ) were use to etet uplite Western lots. A n (rrow in pnel ) is etete y WesternBright Quntum in rief exposure, while muh longer exposure is neee to etet the sme n with SuperSignl West Pio (). SuperSignl West Pio 15 min exposure Pge 3 of 5

sensitive thn ECL Plus in eteting STAT-1 (Figure 6g), n twie s sensitive s ECL in eteting tin (Figure 6). 5.00 µg 2.50 µg tin 1.25 g 625 ng 313 ng 156 ng 78 ng 5.00 µg 2.50 µg STAT-1 1.

4 sensitive thn ECL Plus in eteting STAT-1 (Figure 6g), n twie s sensitive s ECL in eteting tin (Figure 6) µg 2.50 µg tin 1.25 g 625 ng 313 ng 156 ng 78 ng 5.00 µg 2.50 µg STAT µg 625 ng 313 ng 156 ng 78 ng Simply the est hoie for CCD imging ECL WesternBright Quntum outperforms other enhne hemiluminesent regents, proviing superior sensitivity, signl urtion, n liner ynmi rnge. Speilly formulte for CCD imging, WesternBright Quntum mintins etetle signl in two-minute exposure for t lest 10 hours, long fter the signls from other sustrtes hve eye to levels unetetle without exposures mny times longer. e f g ECL Plus WesternBright Quntum Methos Gel eletrophoresis Proteins were seprte on self-me 12 % polyrylmie gels using Lemmli uffer system. Gels were 10 m wie n 0.8 mm thik. Trnsfer Proteins were trnsferre to PVDF memrne using tnk (Ie Sientifi) n uffers evelope y Bolt n Mhoney (1). Trnsfers were onute for 25 min t 24 V. Bloking, Primry n Seonry Antioies, n Wshing Memrnes were loke with 2% non-ft ry milk in AvnWsh uffer for 1 hour t room temperture (RT). Primry n seonry ntioies were ilute s esrie in Tle 2 in loking uffer, n pplie to memrnes for 1 hour t RT. Wshing ws onute with AvnWsh uffer oring to the WesternBright user mnul. Sustrte inution After wshing, lots were inute with hemiluminesent sustrtes s reommene y mnufturers. Inution with WesternBright Quntum sustrte ws one for 2 minutes. Imging After inution, lots were ple on plsti sheet, overe with Srn wrp, n imge using FluorChem Q (Cell Biosienes). Imges were nlyze using AlphView softwre (Cell Biosienes). Figure 6. Detet high n low unne proteins with WesternBright Quntum. Ientil lots ontining seril ilutions of ell A431 lystes were proe with mixture or primry ntioies to tin n STAT-1. The lots were then etete with WesternBright Quntum (pnels n f) or one of two other hemiluminesent sustrtes n imge simultneously with 2 minute exposure using CCD imger. The intensities of the ns were plotte ginst totl protein loe (pnels n g), emonstrting tht WesternBright Quntum provie the highest sensitivity n the roest liner ynmi rnge for oth the high n low unne proteins. Experiment Liner ynmi rnge Signl urtion Cell lystes Primry ntioy Rit ntitrnsferrin (Am, t. no ) nti-gapdh (Millipore, t. no. MAB374) nti-tin (Sigm, t. no. A4700) nti-stat 1 (BD, t. no ) Dilution Tle 2. Antioies n ilutions use. Seonry ntioy 1:10,000 HRP-Gotnti-rit (Avnst, t. no. R ) 1:2,000 HRP-Gotnti-mouse (Avnst, t. 1:1,000 HRP-Gotnti-mouse (Avnst, t. 1:250 HRP-Gotnti-mouse (Avnst, t. Dilution 1:20,000 Pge 4 of 5

5 Referenes 1. Bolt M.W. n Mhoney P.A Anl Biohem Copyright 2010 Avnst. All rights reserve. The Avnst logo is registere tremrks of the Compny. WesternBright Quntum, AvnBlok, AvnWsh n LuentBlue re tremrks of the Compny. All other tremrks, servie mrks n trenmes ppering in this rohure re the property of their respetive owners. Ctlog Numer Prout Size K C20 WesternBright Quntum Western Blotting HRP Sustrte Tril kit size 20 ml (200 m 2 ) K D10 WesternBright Quntum Western Blotting HRP Sustrte (for 1000 m 2 memrne) 100 ml (1000 m 2 ) K D20 WesternBright Quntum Western Blotting HRP Sustrte 200 ml (2000 m 2 ) (for 2000 m 2 memrne) L LuentBlue X-ry film 100 sheets R D50 AvnWsh 10x wshing solution 500 ml R Got-nti-rit HRP-onjugte seonry ntioy 500 μl R Got-nti-mouse HRP-onjugte seonry ntioy 500 μl L Low Fluoresene Western Memrne (PVDF) 7x9 m 10 sheets L Nitroellulose Trnsfer Memrne 0.45 μm 7x9 m 10 sheets L Nitroellulose Trnsfer Memrne 0.22 μm 7x9 m 10 sheets D Avnst Corportion 1505 Ams Drive, Suite B1 Menlo Prk, CA Tel: Fx: Emil: sles@vnst.om Prout informtion: Pge 5 of 5

Title of Experiment: Author, Institute and address:

Title of Experiment: Author, Institute and address: Title of Experiment: Trsfetion of murine mrophge RAW264.7 ells with METAFECTENE PRO. Author, Institute n ress: Ptrizi Pellegtti n Frneso Di Virgilio. Deprtment of Experimentl n Dignosti Meiine, Setion