mcherry Rat Monoclonal Antibody

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1 mcherry Rat Monoclonal Antibody Catalog no. M11217 Table 1 Contents and storage Material Amount Concentration Storage Stability mcherry Rat Monoclonal Antibody, unconjugated 100 μl 2 mg/ml in 1X PBS, 0.09% sodium azide 2 8 C When stored as directed, the product is stable for at least 12 months from the date of receipt. Introduction This product is a rat purified monoclonal antibody against the mcherry Fluorescent Protein. To create this antibody, the full length protein was used as the immunogen in a rat hybridoma cell line to produce a monoclonal IgG2a isotype antibody that detects denatured and native forms of mcherry. Because of its improved brightness, superior photostability, and extremely rapid maturation rate, the mcherry monomeric red fluorescent protein is rapidly becoming the preferred red monomer fluorescent protein for monitoring physiological processes and detecting transgenic expression. This affinity purified rat monoclonal antibody can be used to detect native and denatured forms of mcherry or mcherry fusion proteins in Western analysis, immunoprecipitation, immunocytochemistry, and flow cytometry applications. In Western analysis, the mcherry Rat monoclonal antibody demonstrates no cross reactivity to Emerald GFP, TagGFP, mkate2, or TagRFP (Figure 1, page 2). The same degree of specificity is also demonstrated in immunoprecipitation (IPP), immunocytochemistry (ICC), and flow cytometry applications (Figure 2, page 2). The mcherry Rat monoclonal antibody is supplied at a concentration of 2 mg/ml, total volume of 100 μl, in a solution of 1X PBS and 0.09% sodium azide. It is tested for specificity using Western blot analysis. For Research Use Only. Not for use in diagnostic procedures. MAN Revision: 1.0

Figure 1 Western analysis using the mcherry Rat monoclonal antibody. Lanes 1 and 10: Novex Sharp Pre-Stained Protein Standards (Cat. no.

Emerald GFP (Lane 5), untargeted mcherry (Lane 6 and 7) and Control (Lanes 8 and 9). (A) Rabbit anti-gfp (Cat. no.

R10367) with Alexa Fluor 647 secondary kda 1 2 3 4 5 6 7 8 9 10 kda 1 2 3 4 5 6 7 8 9 10 kda 1 2 3 4 5 6 7 8 9 10 60 60 60 40 40 40 30 30 30 Figure 2 Flow cytometry using the mcherry Rat monoclonal

2 Figure 1 Western analysis using the mcherry Rat monoclonal antibody. Lanes 1 and 10: Novex Sharp Pre-Stained Protein Standards (Cat. no. LC5800), Lanes 2 9: 20 μg of U2OS cell lysate expressing plasma membrane targeted TagRFP (Lane 2), mkate RFP-P62 fusion protein (Lane 3), TagGFP-P62 fusion protein (Lane 4), plasma membrane targeted Emerald GFP (Lane 5), untargeted mcherry (Lane 6 and 7) and Control (Lanes 8 and 9). (A) Rabbit anti-gfp (Cat. no. A11122) with Alexa Fluor 647 secondary (B) Rat anti-mcherry with Alexa Fluor 647 secondary (C) Rabbit anti-rfp (Cat. no. R10367) with Alexa Fluor 647 secondary kda kda kda Figure 2 Flow cytometry using the mcherry Rat monoclonal antibody. U20S cells expressing mcherry were analyzed using 405 nm excitation and 450/40 nm band pass emission on an Attune Acoustic Focusing Cytometer (Life Technologies, Carlsbad, CA). The histogram shows cells stained with mcherry rat monoclonal antibody conjugated with Pacific Blue fluorophore (black line) and unstained cells (gray line). Note: Flow cytometric data shown may not necessarily have been generated using the enclosed lot of reagent. For this reason, and due to differences in flow cytometers and cytometer settings, results may vary from those illustrated above. It is suggested that investigators titrate reagents to determine optimal conditions for use in their systems. mcherry Rat Monoclonal Antibody 2

3 Western Detection Use the following western detection protocol with the anti-mcherry antibody. Be sure to use enough solution in an appropriate container to completely cover the transfer membrane with the solution. Do not allow the membrane to fold or bend. Do not allow any part of the membrane to dry out during the western protocol. Note: It is a good practice to centrifuge the solution briefly in a microcentrifuge before use; add the supernatant to the experiment. This step eliminates any protein aggregates that may have formed during storage and reduces nonspecific background staining. Materials Required but Not Provided 1X Phosphate-buffered saline (PBS, Cat. no ) Tris buffered saline with 0.05% Tween -20 (TBST) Conjugated anti-rat IgG antibody Blocking buffer: 1:1 SeaBlock:TBST plus 0.025% Tween -20 Transfer membrane (nitrocellulose or PVDF) Orbital shaker platform Trays Western Detection Protocol 1. 1 After transferring the proteins to the nitrocellulose or PVDF membrane, rinse the membrane once with PBS. Note: If the PVDF membrane is dry, place the PVDF membrane in 100% methanol for 30 seconds and then place the membrane in TBST for 1 minute. Decant the TBST Place the membrane in the appropriate volume of Blocking buffer in a plastic dish. Incubate the membrane for 1 hour at room temperature on a shaker with gentle agitation. Decant the Blocking buffer Wash the membrane twice with TBST with gentle agitation for 1 minute each Prepare a 1:1000 dilution of the anti-mcherry antibody in Blocking Buffer to obtain a final antibody concentration of 2 μg/ml Decant the TBST and add the diluted anti-mcherry antibody solution from step 1.4 to the membrane. Incubate the membrane for 1 hour at room temperature on a shaker with gentle agitation. Decant the antibody solution Wash the membrane three times with TBST with gentle agitation for 1 minute each Prepare the appropriate conjugated secondary antibody in TBST or blocking buffer according to the manufacturer s recommendations Decant the TBST. Add the diluted secondary antibody solution to the membrane and incubate for 1 hour at room temperature on a shaker with gentle agitation. Decant the antibody solution Wash the membrane three times with TBST with gentle agitation for 5 10 minutes each Continue processing the blot using the appropriate method for the type of conjugate used (e.g., horseradish peroxidase or alkaline phosphatase) until the blot is ready for imaging and detection. mcherry Rat Monoclonal Antibody 3

4 Immunocytochemistry The following protocol is designed for immunocytochemistry using the rat anti mcherry monoclonal antibody. Read the entire protocol before starting. Materials Required but Not Provided Fixative solution: 4% Formaldehyde solution in PBS, ph 7.4 Permeabilizing solution: 0.25% Triton X-100 in PBS, ph 7.4 Blocking solution: 5% Normal Goat Serum (NGS), 3% BSA in PBS, ph 7.4 Conjugated secondary anti-rat IgG antibody for detection 1X Phosphate-buffered saline (PBS) ph 7.4 (Cat. no ) Preparing Cells Culture mammalian cells on cover slips in the appropriate medium to ~75% confluency. Immunocytochemistry Protocol 2. 1 Remove the media from the cells grown on cover slips. Rinse the cells twice for 1 minute each in PBS Fix the cells in Fixative solution (4% formaldehyde in PBS) for 15 minutes at room temperature with gentle agitation in the dark. Remove the solution Wash the cells three times in PBS for 1 minute each with gentle agitation. Remove the PBS Permeabilize the specimen with Permeabilization solution (0.25% Triton X-100 in PBS) for 15 minutes at room temperature with gentle agitation in the dark. Remove the solution Wash the cells three times in PBS for 1 minute each with gentle agitation. Remove the PBS Add Blocking solution (5% Normal Goat Serum, 3% BSA in PBS, ph 7.4) to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution Wash the cells twice in PBS for 1 minute each with gentle agitation Prepare a 1:1000 dilution of Rat anti-mcherry antibody in Blocking buffer to obtain a final antibody concentration of 2 μg/ml Remove the PBS and add the diluted primary antibody solution to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution Wash the cells three in PBS for 1 minute each with gentle agitation Prepare the appropriate conjugated secondary antibody in PBS according to the manufacturer s recommendations Remove the PBS and add the diluted secondary antibody solution to the cells. Incubate for 1 hour at room temperature with gentle agitation. Remove the solution Wash the cells twice in PBS for 2 minutes each with gentle agitation. After the final wash, add PBS to the sample or mount on slides. The sample is now ready for imaging and detection using an appropriate method of choice. mcherry Rat Monoclonal Antibody 4

5 Immunoprecipitation Use the following immunoprecipitation protocol with rat anti-mcherry antibody. Read the entire protocol before starting. Materials Required but Not Provided 1X Phosphate-buffered saline, ph 7.4 (PBS) Cat. no ) Novex Immunoprecipitation Kit Dynabeads Protein G (Cat. no D) Magnetic rack Preparing Magnetic Beads 3. 1 Resuspend Dynabeads magnetic beads in the vial (vortex >30 seconds or tilt and rotate 5 minutes) Transfer 50 μl (1.5 mg) Dynabeads magnetic beads to a tube Place the tube on the magnet to separate the beads from the solution, and remove the supernatant Remove the tube from the magnet. Do not allow the beads to dry out. Immunoprecipitation Protocol 3. 5 Add 1 10 μg of rat anti-mcherry diluted in 200 μl PBS with Tween -20 ( %), to the tube from step 3.4 above Incubate with rotation for 10 minutes at room temperature Place the tube on the magnet and remove the supernatant Remove the tube from the magnet and resuspend the beads-antibody complex in 200 μl PBS with Tween -20. Wash by gentle pipetting. For storing antibody-conjugated Dynabeads magnetic beads, use PBS (ph 7.4) with % Tween -20 to prevent aggregation Place the tube containing Dynabeads magnetic beads-rat anti-mcherry complex on the magnet and remove the supernatant Add your sample containing the mcherry (typically μl) and gently pipette to resuspend the Dynabeads magnetic beads-rat anti-mcherry complex Incubate with rotation for 10 minutes at room temperature to allow antigen to bind to the Dynabeads magnetic beads-antibody complex Place the tube on the magnet. Transfer the supernatant to a clean tube for further analysis, if desired Wash the Dynabeads magnetic beads-rat anti-mcherry complex 3 times using 200 μl washing buffer for each wash. Separate on the magnet between each wash, remove the supernatant, and resuspend by gentle pipetting Resuspend the Dynabeads magnetic beads-rat anti-mcherry complex in 100 μl washing buffer and transfer the bead suspension to a clean tube. This is recommended to avoid co-elution of proteins bound to the tube wall Elute the mcherry using either the Denaturing Elution protocol or the Non denaturing Elution protocol from the Immunoprecipitation Kit-Dynabeads Protein G product information sheet. mcherry Rat Monoclonal Antibody 5

6 Product List Current prices may be obtained from our website or from our Customer Service Department. Cat. no. Product Name Unit Size M11217 mcherry, Rat Monoclonal Antibody, unconjugated μl Purchaser Notification Manufacturing Site 7335 Executive Way Frederick, MD USA Corporate Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone: Fax: European Headquarters Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF UK Phone: Toll-Free Phone: Toll-Free Tech: Fax: Tech Fax: euroinfo@invitrogen.com Tech: eurotech@invitrogen.com Japanese Headquarters LOOP-X Bldg. 6F , Kaigan Minato-ku, Tokyo Japan Phone: Fax: jpinfo@invitrogen.com Additional international offices are listed at These high-quality reagents and materials must be used by, or directl y under the super vision of, a tech nically qualified individual experienced in handling potentially hazardous chemicals. Read the Safety Data Sheet provided for each product; other regulatory considerations may apply. Obtaining Support For the latest services and support information for all locations, go to At the website, you can: Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support (techsupport@lifetech.com) Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents Obtain information about customer training Download software updates and patches SDS Safety Data Sheets (SDSs) are available at Certificate of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Go to and search for the Certificate of Analysis by product lot number, which is printed on the product packaging (tube, pouch, or box). Disclaimer LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. Limited Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at If you have any questions, please contact Life Technologies at Limited Use Label License: Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial services of any kind, including, without limitation, reporting the results of purchaser s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California The trademarks mentioned herein are the property of Life Technologies Corporation and/or their affiliate(s) or their respective owners. Triton is a registered trademark of Union Carbide Corporation. Tween is a registered trademark of Uniqema Americas, LLC Life Technologies Corporation. All rights reserved. 6 May 2013

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