WesternBright TM MCF and MCF-IR

Transcription

1 WesternBright TM MCF nd MCF-IR Quntittive, multi-color fluorescent Western lotting kits WesternBright MCF visile nd ner infrred (IR) fluorescent Western lotting kits llow the ssy of two proteins t once, incresing the qulity nd quntity of informtion tht cn e gined from single lot. Assy control longside protein of interest. Assy phosphorylted nd un-phosphorylted isoforms of protein simultneously. The fluorescent dyes provided with WesternBright MCF nd WesternBright MCF-IR outperform Cy dyes nd llow detection of low pg of protein. The WesternBright MCF protocols sve time nd money since there is no need for disposle film, nd the lot cn e imged immeditely, without drying.

2 WesternBright MCF nd MCF-IR llow the ssy of two or more proteins on one lot with the sensitivity of chemiluminescence. Sve time nd money reltive to chemiluminescence detection y voiding the need to run duplicte lots or to strip nd re-proe lot to study multiple trgets. WesternBright MCF nd MCF-IR re quick, esy to use kits tht provide everything necessry to conduct two-color Westerns. Two-Color Western Blotting The principle of two-color Western lotting. Using secondry ntiodies leled with different fluorophores, two proteins cn e detected on single lot y controlling excittion nd detection chnnels (). WesternBright MCF conjugtes incorporte the phycoiliproteins llophycocynin (APC) nd R-phycoerythrin (RPE), extremely right fluorescent proteins from cynocteri nd eukryotic lge (,c). WesternBright MCF-IR conjugtes incorporte IR700 nd IR800 fluorescent dyes with ner-infrred excittion nd emission wvelengths (d,e). secondry ntiody conjugte secondry ntiody conjugte APC conjugted lgg Dye 1 Dye 2 APC sorption APC emission PVDF memrne primry ntiody (mouse) primry ntiody (rit) Asorption Fluorescence Emission proteins trnsferred to the memrne locking compound RPE sorption sorption RPE emission emission Asorption Fluorescence Emission Wvelength (nm) c RPE conjugted lgg d IR700 conjugted lgg e IR800 conjugted lgg Wvelength (nm)

3 WesternBright TM MCF Brighter thn Cy Dyes WesternBright conjugtes provide righter signl thn the ECL Plex Western lotting system. Duplicte Western lots contining smples of AFP nd CEA proteins were treted identiclly, nd proed with the sme mixture of mouse nti-afp nd rit nti-cea primry ntiodies. One lot ws then stined with WesternBright conjugtes nd the other with n identicl concentrtion of Cy3 nti-mouse nd Cy5 nti-rit ntiodies following the protocol recommended for ECL Plex. Under identicl imging conditions, WesternBright MCF provides righter signl, nd 10x greter sensitivity. Picogrm Detection Levels, Liner Dt 4.65 ng Bnd Intensity 2.33 ng 1.16 ng 581 pg pg 145 pg 73 pg 36 pg 18 pg 9 pg 4.5 pg 2.3 pg R 2 = R² = pg 0.6 pg Detect picogrms of protein with WesternBright MCF nd MCF-IR. WesternBright MCF nd MCF-IR re s sensitive s chemiluminescence, while providing more quntittive dt thn chemiluminescence. The fluorescent signl is directly relted to the mount of ound ntiody, nd not dependnt on n enzymtic rection s is chemiluminescent signl. A lot contining seril dilution of Trnsferrin ws proed with rit-nti-trnsferrin primry ntiody, nd detected using WesternBright conjugte APC-got-nti-rit IgG. As little s 1.1 pg of trnsferrin cn e visulized in the resulting imge (). The signl is liner with respect to protein concentrtion over 3 orders of mgnitude () Trnsferrin (pg)

4 Why Two-Color Westerns? Simultneously Detect Closely Migrting Proteins c Simultneous detection of EGFR nd phospho-egfr with WesternBright MCF. The increse in phosphoryltion of EGFR in response to EGF ws detected. Chemiluminescence detection of the sme experiment would require stripping nd reproing the lot, which cn introduce errors due to protein loss, or incomplete stripping of primry ntiody. Lystes from A431 cells (lne 1) nd from A431 cells treted with EGF (lne 2) were lotted nd EGFR detected in the green chnnel () or phospho-egfr detected in the red chnnel (c). The two chnnels re superimposed in (). Lne 3: moleculr weight mrkers. Get More Dt from Every Blot Multicolor fluorescent WesternBright lots provide quntittive dt. With multicolor fluorescent Westerns, simultneous detection of loding controls llows protein mounts to e ccurtely quntified, nd different smples to e ccurtely compred. WesternBright conjugtes re comptile with common lue dyes, llowing 3-color lots to e conducted. In the figure, Cy2- leled primry ntiody ws used to detect the loding control, trnsferrin, while WesternBright conjugtes were used to detect CEA (red) nd AFP (green) proteins. The intensities of the CEA nd AFP nds, normlized to the loding control, re shown (). Bnd Intensity, normlized to Trnsferrin CEA AFP CEA (APC-Western Bright conjugte) Trnsferrin (Cy2) AFP (RPE- WesternBright conjugte) Lne numer

5 WesternBright TM MCF-IR Two-Color Ner Infrred Blots Detect two proteins in one experiment with WesternBright MCF-IR. Ner IR dyes re idel for Western lot pplictions ecuse utofluorescence from memrnes decrese with wvelengths ner- IR spectr giving you clener western lots. A lot of seril dilutions of trnsferrin nd GAPDH ws proed simultneously with rit-nti-trnsferrin primry ntiody nd mouse-nti-gapdh nd detected with WesternBright got-nti-rit IgG IR700 nd WesternBright got-nti-mouse IgG IR800. kda ! Trnsferrin (78 kd) IR 700! GAPDH (37kD) IR 800 Brod Liner Rnge Bnd Intensity 167 ng 55.6 ng ng 6.18 ng 2 ng 0.68 ng 0.22 ng R 2 = ng Choose WesternBright MCF nd MCF-IR for quntittive lotting experiments. Both kits provide sensitive detection nd rod liner rnge. A lot contining seril dilution of Trnsferrin ws proed with rit-nti-trnsferrin primry ntiody, nd detected using WesternBright got-ntirit IgG IR700. As little s 25 pg of trnsferrin cn e visulized in the resulting imge without sturtion of high concentrtion nd contining 55 ng of protein (). The signl is liner with respect to protein concentrtion over 3 orders of mgnitude () Trnsferrin (ng)

6 WesternBright TM MCF nd MCF-IR Quntittive, multi-color fluorescent Western lotting kits WesternBright MCF nd MCF-IR Western lotting kits provide ll mterils needed to conduct 2-color Western lots: fluorescent leled secondry ntiodies, locking solution, wshing solution, pre-cut low-utofluorescence PVDF memrnes, nd ckground quenching sheets. Fetures FLUORESCENT DETECTION of two proteins, with sensitivity 10x greter thn Cy dyes ADVANBLOCK -PF locking solution for fst, proteinfree locking nd greter signl-to-noise rtios HIGH SENSITIVITY detect picogrms of protein FAST RESULTS entire protocol cn e completed in 3.5 hours WESTERNBRIGHT MCF comptile with imging systems tht detect Cy3 nd Cy5 or similr dyes WESTERNBRIGHT MCF-IR comptile with ner-ir imging systems ADVANWASH nd ADVANWASH-IR wshing solutions optimized for chemiluminescent nd fluorescent or for ner-ir fluorescent Western lots Ordering Informtion Ctlog Numer Product Size K WesternBright MCF fluorescent Western lotting kit Got-nti-rit IgG APC/ Got-nti-mouse IgG RPE K WesternBright MCF-IR fluorescent Western lotting kit Got-nti-rit IgG IR700/ Got-nti-mouse IgG IR800 K WesternBright MCF-IR fluorescent Western lotting kit Got-nti-mouse IgG IR700/ Got-nti-rit IgG IR800 Relted Products 10 lots 10 lots 10 lots R D25 FLASHBlot Trnsfer Buffer, 50x concentrte Sufficient for up to 50 lots 250 ml R D50 FLASHBlot Trnsfer Buffer, 50x concentrte Sufficient for up to 100 lots 500 ml L Pre-cut Blotting Pper 7 x 9 cm, verticl gels 60 sheets L Pre-cut Blotting Pper 8 x 10 cm, verticl gels 60 sheets Advnst Corportion 1505 Adms Drive, Suite B1 Menlo Prk, CA Tel: Fx: Emil: sles@dvnst.com Product informtion: Copyright Advnst. All rights reserved. The Advnst logo is registered trdemrk of the Compny. WesternBright, AdvnBlock nd AdvnWsh re trdemrks of the Compny. All other trdemrks, service mrks nd trdenmes ppering in this rochure re the property of their respective owners. D