Goat Anti Mouse IgG Antibodies

Transcription

1 Goat Anti Mouse IgG Antibodies Table 1. Contents and Storage Information. Material Amount Concentration Storage Upon Receipt Stability Whole antibodies F(ab ) 2 Fragments 0.5 ml 250 μl 2 mg/ml in 0.1 M sodium phosphate, 0.1 M NaCl, 5 mm sodium azide, ph C Protect from light Avoid freeze-thaw cycles When stored undiluted as directed, products are stable for at least 3 months. For longer storage, divide solution into single-use aliquots and freeze at 20 C, which are stable for at least 6 months. R-phycoerythrin (R-PE) conjugates Allophycocyanin (APC) conjugates * Alexa Fluor dye R-PE and APC tandem conjugates 1 ml 0.5 ml 100 μl 1 mg/ml in 0.1 M sodium phosphate, 0.1 M NaCl, 5 mm sodium azide, ph mg/ml in 0.1 M sodium phosphate, 0.1 M NaCl, 2 mm EDTA, 1% glycerol, 5 mm sodium azide, ph C Protect from light DO NOT FREEZE When stored undiluted as directed, products are stable for at least 3 months. BODIPY FL whole antibody conjugates 1 mg Lyophilized powder from 0.1 M sodium phosphate, 0.1 M NaCl, 1.5% bovine serum albumin, 0.01% thimerosal, ph C Desiccate Protect from light Avoid freeze-thaw cycles When stored as directed, products are stable for at least 6 months. * APC conjugates are prepared from chemically crosslinked APC to avoid dissociation of the molecule into subunits when highly diluted. 1 May also contain 1% Prionex reagent as a stabilizer. Spectral data: See Tables 2 and 3. Introduction Invitrogen offers an extensive line of goat anti mouse IgG conjugates labeled with a wide selection of premium fluorescent dyes or with biotin (Table 2). We also offer goat anti mouse IgG conjugated with fluorescent phyco biliproteins, R-phycoerythrin (R-PE) or allophycocyanin (APC), or with phycobiliprotein dye tandem constructs 2 (Table 3). Fluorescent anti mouse IgG conjugates are ideal for fluorescence microscopy and confocal laser scanning microscopy, as well as for flow cytometry. In addition to conjugates of whole IgG antibodies, conjugates of F(ab ) 2 fragments and highly cross-adsorbed whole antibodies are available in several fluorescent colors. Molecular Probes strict quality control procedures and many years of experience in labeling antibodies guarantee that each conjugate provides optimal fluorescence. Revised: 23 February 2009 MP 00852

2 In addition to the antibodies listed in this manual, Invitrogen prepares fluorescent conjugates of many other species-specific anti-igg antibodies, as well as conjugates of avidin, streptavidin, NeutrAvidin biotin-binding protein, protein A, and protein G. For details, visit our website at probes.invitrogen.com or contact our Technical Support Department. Whole Antibody Conjugates The goat anti mouse IgG whole antibody conjugates are prepared from affinity-purified antibodies that react with IgG heavy chains and all classes of immunoglobulin light chains from mouse. To minimize cross-reactivity, the goat anti mouse IgG whole antibodies have been adsorbed against human IgG and human serum prior to conjugation. The degree of labeling for each conjugate is typically 2 8 fluorophore or biotin molecules per IgG molecule; the exact degree of labeling is indicated on the product label. At the time of preparation, the products are certified to be free of unconjugated dyes and are tested in a cytological experiment to ensure low nonspecific staining. F(ab') 2 Fragment Conjugates Conjugates of F(ab') 2 fragments are sometimes preferable to whole antibody conjugates for secondary detection, since the absence of the Fc region in F(ab') 2 fragments prevents interactions with Fc receptor bearing membranes. The F(ab') 2 fragments are prepared from antibodies that have been adsorbed against human IgG and serum to minimize cross-reactivity. The degree of labeling for each conjugate is typically 2 6 fluorophore or biotin molecules per F(ab') 2 fragment; the exact degree of labeling is indicated on the product label. Highly Cross-Adsorbed Whole Antibody Conjugates For researchers interested in highly cross-adsorbed antibodies, we provide labeled goat anti mouse IgG whole antibodies that have been adsorbed against bovine IgG, goat IgG, rabbit IgG, rat IgG, human IgG, and human serum. These highly cross-adsorbed antibodies may be useful in multilabeling experiments, or for labeling cells or tissues where nonspecific staining has been a problem. Because our highly cross-adsorbed antibodies have been adsorbed against rat IgG, they are particularly useful for detecting mouse IgG in rat tissues or cells and in experiments in which antibodies from mouse are being detected in the presence of antibodies from rat. Please note, however, that because rats and mice are closely related, the adsorption against rat IgG may have reduced the specificity of this goat anti mouse IgG antibody preparation for certain mouse IgG subclasses. The degree of labeling for each conjugate is typically 2 8 fluorophore or biotin molecules per IgG molecule; the exact degree of labeling is indicated on the product label. At the time of preparation, the products are certified to be free of unconjugated dyes and are tested in a cytological experiment to ensure low nonspecific staining. Guidelines for Use Preparing BODIPY FL Conjugates After reconstitution with 0.5 ml deionized water, the BODIPY FL product can be stored up to 2 weeks at 2 6 C. For longer storage, divide into single-use aliquots and freeze at 20 C. Frozen aliquots are stable for at least 6 months. Using Conjugate Solutions Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step eliminates any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining. Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. For the fluorophore- and biotin-labeled antibodies, including the Goat Anti Mouse IgG Antibodies 2

3 phycoerythrin-, allophycocyanin-, and tandem-labeled antibodies, a final concentration of 1 10 μg/ml should be satisfactory for most immunohistochemical applications. 3 For flow cytometry applications, μg per cells should yield satisfactory results. Table 2. Goat anti mouse IgG antibodies. Label Ex * Em * Whole antibody Highly cross adsorbed F(ab ) 2 fragment Unlabeled NA NA A10535 A10534 Biotin (Nonfluorescent) Conjugates Biotin-XX NA NA B2763 B11027 DSB-X biotin NA NA D20690 D20691 D20692 Fluorescent Dye Conjugates Alexa Fluor A11045 A21049 A11068 Marina Blue M10991 Cascade Blue C962 Pacific Orange P31583 P31586 P31585 Alexa Fluor A31553 Pacific Blue P10993 P31582 P31581 Alexa Fluor A11063 Fluorescein F2761 F11021 Alexa Fluor A11001 A11029 A11017 Oregon Green O6380 O11033 Alexa Fluor A31554 BODIPY FL B2752 Oregon Green O6383 Alexa Fluor A31555 Alexa Fluor A11002 Cy A10521 Tetramethylrhodamine T2762 Alexa Fluor A11003 A11030 A11018 Alexa Fluor A21422 A21424 A21425 Rhodamine Red -X R6393 Alexa Fluor A11004 A11031 A11019 Alexa Fluor A11005 A11032 A11020 Texas Red T862 Texas Red -X T6390 Alexa Fluor A31550 Alexa Fluor A21050 A21052 A21053 Alexa Fluor A31574 A31575 Cy A10524 Alexa Fluor A21235 A21236 A21237 Alexa Fluor A21054 A21055 Alexa Fluor A21057 A21058 A21059 Alexa Fluor A21036 Alexa Fluor A21037 * Approximate fluorescence excitation (Ex) and emission (Em) maxima, in nm, for conjugates. Complete spectra for most of these dyes are available at our website (probes.invitrogen.com). Cross-adsorbed against human IgG and human serum. Whole antibody, cross-adsorbed against bovine IgG, goat IgG, rabbit IgG, human IgG and serum, and rat IgG. Human vision is insensitive to light beyond ~650 nm, and therefore it is not possible to view the fluorescence of these dyes by looking through a conventional fluorescence microscope. NA = Not applicable. Goat Anti Mouse IgG Antibodies 3

4 Table 3. R-Phycoerythrin, allophycocyanin, and tandem conjugates of goat anti mouse IgG antibodies. Label Ex * Em * Cat. no. F(ab ) 2 fragment R-Phycoerythrin (R-PE) and Tandem R-PE Conjugates R-Phycoerythrin 496, 546, P852 A10543 Alexa Fluor 610 R-PE 496, 546, A20980 Alexa Fluor 647 R-PE 496, 546, A20990 Alexa Fluor 680 R-PE 496, 546, A20983 Allophycocyanin (APC) and Tandem-APC Conjugates Allophycocyanin A865 A10539 Alexa Fluor 680 APC A21000 Alexa Fluor 750 APC A21006 *Approximate fluorescence excitation (Ex) and emission (Em) maxima, in nm, for conjugates. Cross-adsorbed against human IgG and human serum. Multiple absorbance peaks. Human vision is insensitive to light beyond ~650 nm, and therefore it is not possible to view the fluorescence of these dyes by looking through a conventional fluorescence microscope. References 1. Cytometry 8, 91 (1987); 2. Our tandem constructs comprise a donor phycobiliprotein, such as R-PE or APC, coupled to a longer-wavelength light emitting fluorescence acceptor. By the process of fluorescence resonance energy transfer (FRET), an energy transfer cascade is established wherein most of the light absorbed by the donor R-PE or APC results in fluorescence of the acceptor dye. This process can be quite efficient, resulting in almost total transfer of energy to the acceptor dye.; 3. Short Protocols in Molecular Biology, 2nd Edition, F.M. Ausubel et al., Eds., John Wiley and Sons (1992) pp Product List Current prices may be obtained from our website or from our Customer Service Department. Cat. no. Product Name Unit Size A11045 Alexa Fluor 350 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21049 Alexa Fluor 350 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A31553 Alexa Fluor 405 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11063 Alexa Fluor 430 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11017 Alexa Fluor 488 F(ab ) 2 A11001 Alexa Fluor 488 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11029 Alexa Fluor 488 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A31554 Alexa Fluor 500 goat anti-mouse IgG (H+L) *2 mg/ml* ml A31555 Alexa Fluor 514 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11002 Alexa Fluor 532 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11018 Alexa Fluor 546 F(ab ) 2 A11003 Alexa Fluor 546 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11030 Alexa Fluor 546 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A21425 Alexa Fluor 555 F(ab ) 2 A21422 Alexa Fluor 555 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21424 Alexa Fluor 555 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A11019 Alexa Fluor 568 F(ab ) 2 A11004 Alexa Fluor 568 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11031 Alexa Fluor 568 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A11020 Alexa Fluor 594 F(ab ) 2 A11005 Alexa Fluor 594 goat anti-mouse IgG (H+L) *2 mg/ml* ml A11032 Alexa Fluor 594 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A31550 Alexa Fluor 610 goat anti-mouse IgG (H+L) *2 mg/ml* ml Goat Anti Mouse IgG Antibodies 4

5 A20980 Alexa Fluor 610 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml* μl A21053 Alexa Fluor 633 F(ab ) 2 A21050 Alexa Fluor 633 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21052 Alexa Fluor 633 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A31574 Alexa Fluor 635 goat anti-mouse IgG (H+L) *2 mg/ml* ml A31575 Alexa Fluor 635 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A21237 Alexa Fluor 647 F(ab ) 2 A21235 Alexa Fluor 647 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21236 Alexa Fluor 647 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A20990 Alexa Fluor 647 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml* μl A21054 Alexa Fluor 660 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21055 Alexa Fluor 660 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A21059 Alexa Fluor 680 F(ab ) 2 A21057 Alexa Fluor 680 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21058 Alexa Fluor 680 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A21000 Alexa Fluor 680 allophycocyanin goat anti-mouse IgG (H+L) *1 mg/ml* μl A20983 Alexa Fluor 680 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml* μl A21036 Alexa Fluor 700 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21037 Alexa Fluor 750 goat anti-mouse IgG (H+L) *2 mg/ml* ml A21006 Alexa Fluor 750 allophycocyanin goat anti-mouse IgG (H+L) *1 mg/ml* μl A865 allophycocyanin, crosslinked, goat anti-mouse IgG (H+L) *1 mg/ml* ml A10539 allophycocyanin, crosslinked, F(ab ) 2 fragment of goat anti-mouse (H+L) *1 mg/ml* μl B11027 biotin-xx F(ab ) 2 fragment of goat anti-mouse IgG (H+L) *2 mg/ml* μl B2763 biotin-xx goat anti-mouse IgG (H+L) *2 mg/ml* ml B2752 BODIPY FL goat anti-mouse IgG (H+L) mg C962 Cascade Blue goat anti-mouse IgG (H+L) *2 mg/ml* ml A10521 Cy3 goat anti-mouse IgG (H+L) *2 mg/ml* ml A10524 Cy5 goat anti-mouse IgG (H+L) *2 mg/ml* ml D20692 DSB-X biotin F(ab ) 2 fragment of goat anti-mouse IgG (H+L) *2 mg/ml* μl D20690 DSB-X biotin goat anti-mouse IgG (H+L) *2 mg/ml* ml D20691 DSB-X biotin goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml A10534 F(ab ) 2 fragment of goat anti-mouse IgG (H+L) *2 mg/ml* μl F11021 fluorescein F(ab ) 2 fragment of goat anti-mouse IgG (H+L) *2 mg/ml* μl F2761 fluorescein goat anti-mouse IgG (H+L) *2 mg/ml* ml A10535 goat anti-mouse IgG (H+L) *2 mg/ml* ml G21061 goat anti-mouse IgG (H+L), CMNB-caged fluorescein conjugate *2 mg/ml* μl M10991 Marina Blue goat anti-mouse IgG (H+L) *2 mg/ml* ml O6380 Oregon Green 488 goat anti-mouse IgG (H+L) *2 mg/ml* ml O11033 Oregon Green 488 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml O6383 Oregon Green 514 goat anti-mouse IgG (H+L) *2 mg/ml* ml P31581 Pacific Blue F(ab ) 2 fragment of goat anti-mouse IgG (H+L) μl P10993 Pacific Blue goat anti-mouse IgG (H+L) *2 mg/ml* ml P31582 Pacific Blue goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml P31585 Pacific Orange F(ab ) 2 fragment of goat anti-mouse IgG (H+L) *2 mg/ml* μl P31583 Pacific Orange goat anti-mouse IgG (H+L) *2 mg/ml* ml P31586 Pacific Orange goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/ml* ml R6393 Rhodamine Red -X goat anti-mouse IgG (H+L) *2 mg/ml* ml A10543 R-phycoerythrin F(ab ) 2 fragment of goat anti-mouse IgG (H+L) *1 mg/ml* μl P852 R-phycoerythrin goat anti-mouse IgG (H+L) *1 mg/ml* ml T2762 tetramethylrhodamine goat anti-mouse IgG (H+L) *2 mg/ml* ml T862 Texas Red goat anti-mouse IgG (H+L) *2 mg/ml* ml T6390 Texas Red -X goat anti-mouse IgG (H+L) *2 mg/ml* ml Goat Anti Mouse IgG Antibodies 5

6 Contact Information Molecular Probes, Inc Willow Creek Road Eugene, OR Phone: (541) Fax: (541) Customer Service: 6:00 am to 4:30 pm (Pacific Time) Phone: (541) Fax: (541) Toll-Free Ordering for USA: Order Phone: (800) Order Fax: (800) Technical Service: 8:00 am to 4:00 pm (Pacific Time) Phone: (541) Toll-Free (800) Fax: (541) Invitrogen European Headquarters Invitrogen, Ltd. Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF, UK Phone: +44 (0) Fax: +44 (0) euroinfo@invitrogen.com Technical Services: eurotech@invitrogen.com Further information on Molecular Probes products, including product bibliographies, is available from your local distributor or directly from Molecular Probes. Customers in Europe, Africa and the Middle East should contact our office in Paisley, United Kingdom. All others should contact our Technical Service Department in Eugene, Oregon. Molecular Probes products are high-quality reagents and materials intended for research pur pos es only. These products must be used by, or directl y under the super vision of, a tech nically qual i fied individual experienced in handling potentially hazardous chemicals. Please read the Material Safety Data Sheet pro vid ed for each prod uct; other regulatory considerations may apply. Limited Use Label License No. 223: Labeling and Detection Technology The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, Willow Creek Road, Eugene, OR 97402, Tel: (541) Fax: (541) Several Molecular Probes products and product applications are covered by U.S. and foreign patents and patents pending. All names contain ing the des ig na tion are reg is tered with the U.S. Patent and Trade mark Office. Copyright 2009, Molecular Probes, Inc. All rights reserved. This information is subject to change without notice. For country-specific contact information, visit Goat Anti Mouse IgG Antibodies 6

7 Product Information Revised: 23 August 2004 Alexa Fluor 568 Protein Labeling Kit (A10238) Storage upon receipt: Component A 20 C Protect from light Protect from moisture Components B 2 6 C Protect from moisture Components C and D 2 6 C only, DO NOT FREEZE COMPONENT C Abs/Em of conjugate: 555/565 nm Introduction Molecular Probes Alexa Fluor 568 Protein Labeling Kit provides a convenient means to label proteins with our superior Alexa Fluor 568 dye. Alexa Fluor 568 dye labeled proteins, which have absorption and fluorescence emission maxima of approximately 577 nm and 603 nm, respectively, are ideal probes for excitation by the 568 nm spectral line of Ar-Kr lasers. The Alexa Fluor 568 Protein Labeling Kit contains everything that is required to perform three separate labeling reactions and to purify the resulting conjugates. The Alexa Fluor 568 reactive dye (Figure 1) has a succinimidyl ester moiety that reacts efficiently with primary amines of proteins to form stable dye CH 3 CH 3 H N CH 2 SO 3 OC O O O N H N + CH 3 CH 3 C OHCH SO 2 3 O protein conjugates. Each of the three vials of reactive dye provided in the kit is sufficient for labeling ~1 mg of an IgG antibody, although other proteins can also be labeled. Materials Contents $ Alexa Fluor 568 reactive dye (Component A), three vials, each containing a magnetic stir bar $ Sodium bicarbonate (MW = 84, Component B), 84 mg $ Purification resin (Component C), ~25 ml $ 10X Elution buffer (Component D), ~25 ml $ Purification columns, three $ Column funnels, three $ Foam column holders, three $ Disposable pipets, three $ Collection tubes, three 4 ml tubes Storage Upon receipt, store Component A at 20 o C and Components B-D at 2 6 o C. DO NOT FREEZE THE PURIFICATION RESIN (Component C). Protect the reactive dye (Component A) from light. The reactive dye and sodium bicarbonate (Component B) should be protected from moisture. When stored properly, the kit components should be stable for at least three months. Protein Preparation IMPORTANT: For optimal labeling efficiency, the purified protein must be in a buffer free of ammonium ions or primary amines. If the protein is in an unsuitable buffer (e.g. Tris or glycine), the buffer should be replaced with phosphate-buffered saline (PBS) by dialysis or another method. Impure proteins (e.g. antibodies in crude serum) will not label well. The presence of low concentrations of sodium azide ( 3 mm) or thimerosal ( 1 mm) will not interfere with the conjugation reaction. This kit can be used to label virtually any protein, although the following protocol has been optimized for labeling IgG antibodies. Each vial of reactive dye contains the appropriate amount of dye to label approximately 1 mg of IgG (MW ~145,000) as 0.5 ml of IgG solution at 2 mg/ml. For tips on optimizing the procedure for other proteins or for antibody solutions at lower concentrations, see Tips for Using the Kit with Other Proteins and/or Concentrations and Troubleshooting. Figure 1. Alexa Fluor 568 carboxylic acid, succinimidyl ester (MW ~792). O MP Alexa Fluor 568 Protein Labeling Kit

8 Labeling Reaction 1.1 Prepare a 1 M solution of sodium bicarbonate by adding 1 ml of deionized water (dh 2 O) to the provided vial of sodium bicarbonate (Component B). Vortex or pipet up and down until fully dissolved. The bicarbonate solution, which will have a ph ~8.3, can be stored at 4 C for up to two weeks. 1.2 If the protein concentration is greater than 2 mg/ml, the protein should be diluted to 2 mg/ml in a suitable buffer, e.g. PBS or 0.1 M sodium bicarbonate. 1.3 To 0.5 ml of the 2 mg/ml protein solution, add 50 µl of 1 M bicarbonate (prepared in step 1.1). Bicarbonate, ph ~8.3, is added to raise the ph of the reaction mixture, since succinimidyl esters react efficiently at ph Allow a vial of reactive dye to warm to room temperature. Transfer the protein solution from step 1.3 to the vial of reactive dye. This vial contains a magnetic stir bar. Cap the vial and invert a few times to fully dissolve the dye. Stir the reaction mixture for 1 hour at room temperature. Because preparation of the purification column takes ~15 minutes, you may wish to begin pouring the column (see Purification of the Labeled Protein) during the labeling reaction. Purification of the Labeled Protein 2.1 Assemble the column and position it upright (see Figure 2): Attach a funnel to the top of a column. Gently insert the column through the X-cut in one the provided foam holders. Using the foam holder, secure the column with a clamp to a ringstand. Carefully remove the cap from the bottom of the column. The foam holder is provided to prevent the clamp from damaging the column. 2.2 Prepare elution buffer by diluting the 10X stock (Component D) 10-fold in dh 2 O. Typically, less than 10 ml will be required for each purification. Set aside until step 2.5. The 10X elution buffer (10X PBS) contains 0.1 M potassium phosphate, 1.5 M NaCl, ph 7.2, with 2 mm sodium azide. The 10X stock should be warmed to room temperature prior to use to ensure that the buffer is fully dissolved. Sufficient elution buffer is included to allow washing of the columns for reuse, if desired. 2.3 Using one of the provided pipets, stir the purification resin (Component C) thoroughly to ensure a homogeneous suspension. Pipet the resin into the column, allowing excess buffer to drain away into a small beaker or other container. Resin should be packed into the column until the resin is ~3 cm from the top of the column. Component C, Bio-Rad BioGel P-30 Fine size exclusion purification resin, is designed to separate free dye from proteins with MW > 40,000. This is packaged in PBS containing 2 mm sodium azide. For smaller proteins, gel filtration media of a suitable molecular weight cutoff should be selected. Labeled peptides may be separated from free dye by TLC or HPLC. 2.4 Allow the excess buffer to drain into the column bed. Do not worry about the column drying out, since the matrix will remain hydrated. Make certain the buffer elutes through the column with a consistently even flow prior to adding the reaction mixture. If the flow of buffer is slow or stalled, repack the column. Carefully load the reaction mixture from step 1.4 onto the column. You may wish to remove the column funnel to load the sample. Allow the mixture to enter the column resin. Rinse the reaction vial with ~100 µl of elution buffer and apply to the column. Allow this solution to enter the column. 2.5 Replace the funnel if it was removed for sample loading. Slowly add elution buffer (prepared in step 2.2), taking care not to disturb the column bed. Continue adding elution buffer until the labeled protein has been eluted (typically about 30 minutes). IMPORTANT: Collect, and retain as fractions, all of the eluted buffer. 2.6 As the column runs, periodically illuminate the column with a handheld UV lamp. You should observe two fluorescent bands, which represent the separation of the labeled protein from the unincorporated dye. Collect the first band, which contains the labeled protein, into one of the provided collection tubes. Add elution buffer to the column as necessary. Do not collect the slower moving band, which consists of unincorporated dye. Once the fraction containing the labeled protein has been successfully collected, all other fractions of eluted buffer may be discarded. In rare instances where there is no discernable band corresponding to labeled protein, the retained fractions can be used to recover any unlabeled protein. Determination of Degree of Labeling Figure 2. Column assembly. 3.1 Measure the absorbance of the conjugate solution at 280 nm and 577 nm (A 280 and A 577 ) in a cuvette with a 1 cm pathlength. Dilution of the sample may be necessary. 2 Alexa Fluor 568 Protein Labeling Kit

9 3.2 Calculate the concentration of protein in the sample: protein concentration (M) [ ] A (A 0.46) dilution factor = 203,000 where 203,000 cm -1 M -1 the molar extinction coefficient of a typical IgG and 0.46 is a correction factor to account for absorption of the dye at 280 nm. Non-IgG proteins will likely have significantly different molar extinction coefficients. 3.3 Calculate the degree of labeling: A 577 dilution factor moles dye per mole protein = 91,300 protein concentration (M) where 91,300 cm -1 M -1 is the approximate molar extinction coefficient of the Alexa Fluor 568 dye at 577 nm. For IgGs, we find that optimal labeling is achieved with 2 6 moles of Alexa Fluor 568 dye per mole of antibody. Storage and Handling of Conjugates Store the labeled protein which will be in PBS, ph 7.2, containing ~2 mm sodium azide at 4 C, protected from light. If the final concentration of purified protein conjugate is less than 1 mg/ml, add bovine serum albumin (BSA) or other stabilizing protein to 1 10 mg/ml. The conjugate should be stable at 4 C for several months. For long-term storage, divide the solution into small aliquots and freeze at 20 C. AVOID REPEATED FREEZING AND THAWING. PROTECT FROM LIGHT. It is a good practice to centrifuge conjugate solutions in a microcentrifuge before use; only the supernatant should then be used in the experiment. This step will remove any aggregates that may have formed during storage. Tips for Using the Kit with Other Proteins and/or Concentrations Proteins at less than 2 mg/ml. Proteins at concentrations less than 2 mg/ml will not label as efficiently. If the protein cannot be concentrated to ~2 mg/ml, you may wish to use less than 1 mg protein per reaction to increase the molar ratio of dye to protein. In addition, using a dilute protein solution, especially at <1 mg/ml, will make it more difficult to efficiently remove the unconjugated dye from the dye-labeled protein with acceptable yields, since the provided purification columns are designed to purify conjugates from a total volume of less than 1 ml. For reaction volumes greater than 1 ml, you can divide the solution of the conjugate and apply it to multiple purification columns or, to avoid further dilution of the conjugate, you can remove free dye by extensive dialysis. Proteins with MW other than ~145,000. Typically, lower MW proteins require fewer dye molecules and higher MW proteins require more dye molecules per protein for optimal labeling. For this reason, we recommend initially performing the reaction with 0.5 ml of 2 mg/ml protein solution, as described for IgGs. The labeling conditions can then be optimized based on the initial results, if desired. Troubleshooting Under-labeling. If calculations indicate that the protein is labeled with significantly less than two moles of fluorophore per mole of a 145,000 dalton protein, your protein may be underlabeled. A number of conditions can cause a protein to label inefficiently: $ Trace amounts of primary amine containing components in the buffer will react with the dye and decrease the efficiency of protein labeling. If your protein has been in amine-containing buffers (e.g. Tris or glycine), dialyze extensively versus PBS before labeling. $ Dilute solutions of protein ( 1 mg/ml) will not label efficiently. Please see step 3.1. $ The addition of sodium bicarbonate (step 1.3) is designed to raise the ph of the reaction mixture to ~8, as succinimidyl esters react most efficiently with primary amines at slightly alkaline ph. If the protein solution is strongly buffered at a lower ph, the addition of bicarbonate will not raise the ph to the optimal level. Either more bicarbonate can be added, or the buffer can be exchanged with PBS, which is only weakly buffered, or with 0.1 M sodium bicarbonate, ph 8.3, by dialysis or other method prior to starting the reaction. $ Because proteins, including different antibodies, react with fluorophores at different rates and retain biological activity at different degrees of dye labeling, the standard protocol may not always result in optimal labeling. To increase the amount of labeling, you can relabel the same protein sample, or you can label a new protein sample using either less protein or more reactive dye per reaction. To increase the amount of dye in the reaction, you can combine the contents of two vials of reactive dye together. Some researchers obtain better labeling with overnight incubations at 4 C after an initial incubation of one hour at room temperature. Over-labeling. If calculations indicate that the protein conjugate is labeled with significantly more than six moles of fluorophore per mole of a 145,000 dalton protein, your protein may be over-labeled. Although conjugates with a high number of attached dye molecules may be acceptable for use, over-labeling can cause aggregation of the protein conjugate and can also reduce the antibody s specificity for its antigen both of which can lead to nonspecific staining. Over-labeling can also cause fluorescence quenching of the attached dyes, which will decrease the fluorescence of the conjugate. To reduce the amount of labeling next time, you can either add more protein to your reaction to decrease the molar ratio of dye to protein or allow the reaction to proceed for a shorter time. Inefficient removal of free dye. Although we have had good success in removing free dye from protein conjugates with the provided columns, it is possible that trace amounts of free dye 3 Alexa Fluor 568 Protein Labeling Kit

10 will remain in the conjugate solution after purification, particularly if a low molecular weight protein is labeled. The presence of free dye, which can be determined by thin layer chromatography, will result in erroneously high calculated values for the degree of labeling (see Determination of Degree of Labeling). Remaining traces of free dye can be removed by applying the conjugate to another column or by extensive dialysis. Product List Current prices may be obtained from our Web site or from our Customer Service Department. Cat # Product Name Unit Size A10238 Alexa Fluor 568 Protein Labeling Kit *3 labelings*... 1 kit Contact Information Further information on Molecular Probes products, including product bibliographies, is available from your local distributor or directly from Molecular Probes. Customers in Europe, Africa and the Middle East should contact our office in Leiden, the Netherlands. All others should contact our Technical Assistance Department in Eugene, Oregon. Please visit our Web site for the most up-to-date information Molecular Probes, Inc Willow Creek Road, Eugene, OR Phone: (541) Fax: (541) Customer Service: 6:00 am to 4:30 pm (Pacific Time) Phone: (541) Fax: (541) order@probes.com Toll-Free Ordering for USA and Canada: Order Phone: (800) Order Fax: (800) Technical Assistance: 8:00 am to 4:00 pm (Pacific Time) Phone: (541) Toll-Free: (800) Fax: (541) tech@probes.com Molecular Probes Europe BV Poortgebouw, Rijnsburgerweg AA Leiden, The Netherlands Phone: Fax: Customer Service: 9:00 to 16:30 (Central European Time) Phone: Fax: eurorder@probes.nl Technical Assistance: 9:00 to 16:30 (Central European Time) Phone: Fax: eurotech@probes.nl Molecular Probes products are high-quality reagents and materials intended for research purposes only. These products must be used by, or directly under the supervision of, a technically qualified individual experienced in handling potentially hazardous chemicals. Please read the Material Safety Data Sheet provided for each product; other regulatory considerations may apply. Several Molecular Probes products and product applications are covered by U.S. and foreign patents and patents pending. Our products are not available for resale or other commercial uses without a specific agreement from Molecular Probes, Inc. We welcome inquiries about licensing the use of our dyes, trademarks or technologies. Please submit inquiries by to busdev@probes.com. All names containing the designation are registered with the U.S. Patent and Trademark Office. Copyright 2004, Molecular Probes, Inc. All rights reserved. This information is subject to change without notice. 4 Alexa Fluor 568 Protein Labeling Kit