Technical Note. One-Blot Western Optimization Using the MPX Blotting System. Aerius, and Odyssey Family of Imaging Systems.

Transcription

1 Techical Note Oe-Blot Wester Optimizatio Usig the MPX Blottig System Developed for: Aerius, ad Odyssey Family of Imagig Systems Please refer to your maual to cofirm that this protocol is appropriate for the applicatios compatible with your Odyssey Imager model. Published May Revised May The most recet versio of this Techical Note is posted at

2 Page 2 Oe-Blot Wester Optimizatio Usig MPX Blottig System Table of Cotets Page I. Itroductio II. Required Reagets III. Gel Electrophoresis & Trasfer IV. Membrae Blockig V. Aligmet i MPX Blotter VI. Primary & Secodary Atibody Applicatio VII. Imagig I. Itroductio The idepedet chaels of the LI-COR MPX (Multiplex) Blotter make it possible to optimize blockig buffer, primary atibody dilutio, ad secodary atibody dilutio o a sigle Wester blot. Wester blottig procedures that geerate a blot of 7.0 x 8.5 cm are easily adapted to the MPX format. The process fits ito ay laboratory s stadard Wester blot workflow. Both homemade ad pre-cast gels ca be used to geerate blots. Electrophoresis ad trasfer to itrocellulose membrae are performed uder stadard coditios. Clampig the blot ito the MPX Blotter creates up to 24 idepedet chaels, allowig differet coditios to be tested i each chael. The rage of usable chaels per sample is relative to comb size. For Wester blot optimizatio, a sigle-well gel ( prep gel ) is all that is eeded. For this applicatio, ay detectio method ca be used, icludig ear-ifrared (NIR) fluorescece ad chemilumiescece. This documet presets geeral guidelies for use with the Odyssey family of Ifrared Imagig Systems. II. Required Reagets LI-COR P/N Sample Preparatio 4X Protei Sample Loadig Buffer Electrophoresis Odyssey Oe-Color Protei Markers (Molecular Weight - 10 kda to 250 kda) Chameleo Duo Pre-staied Protei Ladder (8 kda to 260 kda) Blottig ad Trasfer Tris Glycie Odyssey Nitrocellulose (10 membraes; 7 x 8.5 cm) or Odyssey Nitrocellulose (1 roll; 30 cm x 3 m)

3 Oe-Blot Wester Optimizatio Usig MPX Blottig System Page 3 LI-COR P/N MPX Detectio Blockig Buffer Optimizatio Kit Odyssey Blockig Buffer (TBS) ; Odyssey Blockig Buffer (PBS) ; Casei Blockig Buffer ; Blockig buffer of your choice (BSA, Milk, etc.) IRDye Labeled Secodary Atibodies LI-COR NewBlot IR Strippig Buffer, 5X X PBS 10X TBS Twee 20 MPX Membrae Cushio Imagig Odyssey Family Imager or Aerius Imager III. Gel Electrophoresis ad Trasfer Gel Preparatio A wide variety of gel matrices are compatible with the MPX Blotter system. If you are pourig your ow gels, your gel castig system ca be used with a sigle-well comb such as the LI-COR Sigle Marker/Oe Lae Comb ( , 1 mm thickess). Alteratively, pre-cast gels ca be purchased ad used. Table 1 lists several suggested types of pre-cast gels, ad idicates the umber of usable ports that each gel will provide for use with the MPX Blotter. Table 1. Sigle-sample pre-cast gel optios for use with the MPX Blotter. MW Marker Usable Vedor Well Desigatio Sample # Well Ports Ivitroge 2D 1 Yes 19 Bio-Rad 2D/Prep 1 Yes 21 C.B.S. Scietific 1 Well 1 No 23 Sample Preparatio Whe usig a sigle-well gel, a larger volume of sample is required. Prepare your protei sample so that the sample volume ad cocetratio is equivalet to ruig all the laes o a stadard 10-well gel. Example: 5 µg of lysate per lae = 50 µg i a total volume of µl, icludig loadig buffer.

4 Page 4 Oe-Blot Wester Optimizatio Usig MPX Blottig System The followig procedure is suggested: Dilute the sample 1:4 i 4X Protei Sample Loadig Buffer (LI-COR P/N ) with β-mercaptoethaol. Heat the sample at 95 C for 5 miutes. Molecular Weight Marker It is importat to have a molecular weight marker that is visible to the eye because the marker is the primary tool used to alig the blot i the MPX Blotter. Odyssey Oe-Color Marker or Chameleo Duo Protei Ladder is recommeded. Electrophoresis IMPORTANT: The maximum legth of the separatig gel should ot exceed 50 mm the legth of the chaels o the MPX Blotter. Trasfer Always use clea forceps whe hadlig membraes. Nitrocellulose membrae is recommeded for this procedure. Oce electrophoresis is complete, trasfer proteis to Odyssey Nitrocellulose Membrae usig stadard trasfer procedures. Mark the outside corers of the gel ad sample wells with a pecil before separatig the trasferred gel from the membrae, as show i Figure 1. The marks will help you correctly alig the membrae whe it is placed i the MPX Blotter. Figure 1. Mark the membrae with pecil, for later aligmet ito the MPX Blotter. Allow the membrae to dry for a miimum of oe hour before proceedig with detectio. IMPORTANT! Use pecil to mark the blot. Ik from most pes will fluoresce o the Odyssey Imager ad cause icreased membrae backgroud. IV. Membrae Blockig Membrae Preparatio Cut the membrae ito four idividual blots, as show i Figure 2. Each idividual blot will be processed with a differet blockig buffer, ad that blockig buffer will also be used for dilutio of atibodies. TBS or PBS buffer systems may be used for blockig. Durig washig steps, rise ad wash each blot with a appropriate wash buffer that matches the buffer system used for blockig. Blot 1: Odyssey Blockig Buffer (TBS) Blot 2: Odyssey Blockig Buffer (PBS) Blot 3: Casei Blockig Buffer Blot 4: Blockig buffer of your choice (milk, BSA, etc. i TBS or PBS) Pre-wet each membrae with TBS or PBS buffer as appropriate (see above).

5 Oe-Blot Wester Optimizatio Usig MPX Blottig System Page 5 Figure 2. Cut the membrae ito four idividual blots for blockig buffer optimizatio. Figure 3. Place four idividual blots ito the MPX Blotter as show. Blockig Place the membraes ito 4 differet icubatio boxes. I each box, cover the etire membrae with blockig buffer (approximately 0.4 ml/cm 2 ), usig a differet blockig buffer for each membrae. Block the membrae for 1 hour at room temperature with getle shakig. Blot 1: Blot 2: Blot 3: Blot 4: Odyssey Blockig Buffer (TBS) Odyssey Blockig Buffer (PBS) Casei Blockig Buffer Blockig buffer of your choice (milk, BSA, etc.) V. Aligmet i MPX Blotter Detailed istructios for use of the MPX Blotter are foud i the MPX Blotter Multiplex Wester Blottig Accessory User Guide at Place the four blocked membraes ito the MPX Blotter so that there are at least 4 chaels available for use o each membrae. See Figure 3. VI. Primary & Secodary Atibody Applicatio Primary Atibody Preparatio Two dilutios of primary atibody should be made for each blockig buffer that is tested. Suggested startig dilutios are 1:500 ad 1:1,000. You may wish to modify these dilutios, based o vedor recommedatios.* 700 µl of each dilutio will be eeded. Dilute the primary atibody i the appropriate blockig buffer (see followig table) with 0.2% Twee 20. *The correct workig rage for atibody dilutio depeds o the characteristics of your primary atibody. Start with the dilutio recommeded by the primary atibody vedor for Wester blot applicatios.

6 Page 6 Oe-Blot Wester Optimizatio Usig MPX Blottig System. Blot Blocker Primary Atibody Dilutios 1 Odyssey Blockig Buffer (TBS) 1:500 1:1,000 2 Odyssey Blockig Buffer (PBS) 1:500 1:1,000 3 Casei Blockig Buffer 1:500 1:1,000 4 Blockig buffer of your choice (milk, BSA, etc.) 1:500 1:1,000 Primary Atibody Applicatio Load the primary atibody/blocker dilutios ito the MPX Blotter i the idicated locatios for each blockig buffer you are testig. Apply 2 replicates of each primary atibody dilutio, as show i Figure 4. Fill the uused chaels with the appropriate correspodig blockig buffer. Icubate for 1-4 hours at room temperature. Figure 4. Placemet of primary atibody dilutios i the chaels of the MPX Blotter. Wash primary atibody from the chaels thoroughly accordig to MPX Blotter maual istructios, usig a buffer that matches the buffer system used for blockig. Wash buffers should cotai 0.1% Twee 20. Do ot remove blot from MPX blottig maifold durig washig. Blot 1: Blot 2: Blot 3: Blot 4: TBS-T PBS-T PBS-T TBS-T or PBS-T, as appropriate

7 Oe-Blot Wester Optimizatio Usig MPX Blottig System Page 7 Secodary Atibody Preparatio Blot Blocker Secodary Atibody Dilutios 1 Odyssey Blockig Buffer (TBS) 1:5,000 1:10,000 2 Odyssey Blockig Buffer (PBS) 1:5,000 1:10,000 3 Casei Blockig Buffer 1:5,000 1:10,000 4 Blockig buffer of choice 1:5,000 1:10,000 Two dilutios of secodary atibody should be made for each blockig buffer that is tested. For IRDye secodary atibodies, we recommed 1:5,000 ad 1:10,000 as a startig poit. Dilutios may be modified, based o vedor recommedatios. 700 µl of each atibody will be eeded. Dilute the secodary atibody i the appropriate blockig buffer with 0.2% Twee 20. Secodary Atibody Applicatio Load the secodary atibody/blocker dilutios ito the MPX Blotter i the idicated locatios for each blockig buffer you are testig. Add the secodary atibody dilutios to the chaels previously staied with primary atibody, as show i Figure 5. Fill the uused chaels with the appropriate correspodig blockig buffer. Icubate 1 hour at room temperature. Protect from light durig icubatio. Figure 5. Placemet of secodary atibody dilutios i the chaels of the MPX Blotter.

8 Page 8 Oe-Blot Wester Optimizatio Usig MPX Blottig System VII. Imagig Membraes ca be imaged immediately. Image all four blots side-by-side, usig stadard Wester blot imagig settigs o ay Odyssey Family Imagig System. Visual ispectio of images with Image Studio software or Odyssey applicatio software ca be used to determie which blockig buffer works best for the primary atibody you are testig. View all blots together i a sigle image, with uiform image display settigs, to compare membrae backgroud levels ad bad itesity. Idividually adjust the image display settigs for each blot to get the best image. Evaluate o-specific badig i each blockig buffer coditio. Look for blockig buffer coditios that provide strog sigals for the expected bad(s), low membrae backgroud, ad few o-specific backgroud bads from the primary atibody. Tradeoffs may be ecessary. Blockig coditios that yield very strog bads might also have higher membrae backgroud or o-specific badig. The best blockig coditios deped o the atige-atibody pair you are usig. Some primary atibodies are dramatically affected by blockig coditios. A iappropriate blocker ca alter bidig specificity, affectig the itesity of target bads ad icreasig o-specific badig. The patter of o-specific bads may also be affected. Choose the blockig coditios that are most appropriate for the cotext ad goals of your experimet. Quatitative aalysis of specific bads o each blot will idicate if sigal itesity (after backgroud subtractio) is sigificatly differet betwee blocker types LI-COR, Ic. LI-COR, Odyssey, MPX, Image Studio, Chameleo, NewBlot, ad IRDye are trademarks or registered trademarks of LI-COR, Ic. i the Uited States ad other coutries. All other trademarks belog to their respective owers. LI-COR is a ISO 9001 registered compay Superior Street Licol, NE Toll free: Fax: biosales@licor.com LI-COR GmbH, Germay Servig Europe, Africa, ad the Middle East. LI-COR Bioscieces GmbH Siemesstra e 25A Bad Homburg Germay Phoe: +49 (0) Fax: +49 (0) bio-eu@licor.com LI-COR Ltd., Uited Kigdom Servig Demark, Irelad, Filad, Icelad, Norway, Swede, ad UK. LI-COR Bioscieces UK Ltd. St. Joh s Iovatio Cetre Cowley Road Cambridge CB4 0WS Uited Kigdom Phoe: +44 (0) Fax: +44 (0) bio-eu@licor.com Doc # /16