WakoPureChemical. No WakoPURE system Quick-CBB PLUS Silver Stain MS Kit Negative Gel Stain MS Kit Matrix for MALDI-TOFMS BAMBANKER

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1 WakoPureChemical W P roduct U pdate Protein Research Cell Culture Silver Stain MS Kit Negative Gel Stain MS Kit Matrix for MALDI-TOFMS BAMBANKER No.0 004

2 Index Protein Research. in vitro Protein-Synthesizing System... (#99-90, 9-903) Ni Agarose (#4-0798, , ) WakoPURE MF-00K (#37-03) WakoPURE Spin Empty Column (#34-04). Simple SDS-PAGE Gel Staining... (#74-003, 78-00) Molecular Weight Marker, High Range (#34-40) Molecular Weight Marker, Middle Range (#3-4) 3. Proteome Research... 3 a. Silver Stain MS Kit... 3 Silver Stain MS Kit (#99-890) b. Negative Gel Stain MS Kit... 4 Negative Gel Stain MS Kit (#93-770) c. High purity Matrix at MALDI-TOFMS Analysis... a-cyano-4-hydroxycinnamic Acid [CHCA] (#037-96) Sinapic Acid [SA] (#9-336),-Dihydroxybenzoic Acid [DHB] (#044-90) Lysyl Endopeptidase, MS (#-006) Trypsin, from Porcine Pancreas, MS (#0-9) Cell Culture. Cell Freezing... 6 BAMBANKER TM (#30-468) ALPHABETICAL INDEX A B C D L M N Q S T W page 6 3, 4 3 Ni Agarose BAMBANKER TM CHCA a-cyano-4-hydroxycinnamic Acid,-Dihydroxybenzoic Acid DHB Lysyl Endopeptidase, MS Molecular Weight Marker, High Range Molecular Weight Marker, Middle Range Negative Gel Stain MS Kit Ni Agarose SA Silver Stain II Kit wako Silver Stain MS Kit Sinapic Acid Trypsin, from Porcine Pancreas, MS WakoPURE MF-00K WakoPURE Spin Empty Column INDEX Online Catalog :

3 . in vitro Protein-synthesizing System Next-Generation in vitro Protein-synthesizing System Individual Components bring out the Maximum Performance! is a proprietary protein synthesis & purification technology, which is an in vitro protein synthesizing system "reconstituted" from translation factors expressed in Escherichia coli. It is a novel reconstituted system consisting of around 30 purified enzymes necessary for transcription, translation and energy recycling. All the components involved in transcription and translation except for ribosomes are tagged with hexahistidine at the N or C terminus. Because all the factors are tagged and known components, synthesized protein can be easily purified by simply removing both the ribosome by ultrafiltration and the histidine-tagged factors and enzymes by metal affinity resin. protein of interest can be synthesized and purified about 3 hours. No Tag Required Because all the transcription/translation factors are tagged, synthesized protein can be easily purified. Native form, without any tags. Time Saving All you need is to only 3 steps; synthesis, affinity chromatography and ultrafiltration. It takes only minute for handling and hours for incubation and purification. Pure Protein Synthesis System little contain contaminants such as protease and nuclease because of a novel reconstituted system. Up to 00 Ug protein per ml reaction DHFR (dihydrofolate reductase) can be synthesized at a yield of 0 Ug/mL in hour. Step Step Step 3 3 hour Procedure or Addition of Template DNA* starts transcription/ translation reaction : Template DNA must contain a T7 * promoter sequence. Addition of Ni-Agarose (for adsorption of tagged factors) Removal of ribosome and tagged factors by Ultrafiltration membrane A B Template DNA Ribosome His-Tagged Factors Synthesized protein Metal Affinity Resin M Figure M, 6 : Molecular Weight Marker : Negative Control (no DNA template) : Positive Control (DHFR control plasmid) at Step 3 : Sample obtained at Step was purified by ultrafiltration with WakoPURE MF-00K (Wako Cat. #37-03) 4 : Sample obtained at Step was treated by Ni-Agarose (Wako Cat.#4-0798) (removal of His-tagged factors) and filtered by WakoPURE Spin Empty Column (Wako Cat. #34-04). : Sample obtained at Step was treated by Ni-Agarose and purified by ultrafiltration with WakoPURE MF-00K Result of DHFR after synthesis and various purification. (Apply samples on a.% SDS-PAGE gel and electrophoresed, followed by staining the gel with Silver Stain II Kit Wako (Wako Cat. #9-030) in 3 hours Purified Protein [Note] Plasmid DNA and PCR product can be used with as a template DNA, and the template must contain the following components: An initiation codon (ATG)/A stop codon (TAG, TGA or TAA)/ A T7 promoter sequence located upstream of the target gene/ Prokaryotic Shine-Dalgarno ribosome binding site (SD sequence) located 0 nt upstream of the initiation codon./ More than 6 nt must have at the upstream of the stop codon for the PCR product. No terminator sequence is necessary./ A T7 terminator located in the downstream of the stop codon for plasmid DNA. Kit contents Solution A/ Solution B/DHFR control vector (positive control)/ Universal Primer 4 reactions 6 reactions This kit provides the necessary for procedures until protein synthesis at the step. Related Products () (ml), (0mL), (00mL) (0 EA) (0 EA) Ni-Agarose WakoPURE MF-00K WakoPURE Spin Empty Column ultrafiltration membrane filtration column Available Soon Wako Product Update No.0

4 . Simple SDS-PAGE gel staining Cat.# ml L is a bottled protein staining kit, which allows simple and quick staining of proteins bands in polyacrylamide slab gels. In comparison with conventional Quick- CBB, fixing procedure is not required and organic solvents such as methanol and acetic acid are not used. The mixing procedure is now also unnecessary as all the required solutions for staining are contained in a single bottle. There are also other improvements such as coloration not occurring on the background. As in conventional Quick-CBB, destaining procedure is not required. No fixing procedure and no organic solvents are necessary. Simple and quick staining without the coloration on background Staining Protocol. Washing Wash gel times with deionized water. Staining Stain gel by Wash gel for adding Quick- hour with CBB PLUS 3. Washing deionized water minutes x times Shake for 30 ~ 60 min. Shake for 60 min. FAQ Q : When protein bands are detected a few minutes after the start of staining, can the staining procedure be terminated by washing the gel? A : usually stains protein bands in 0-0 minutes. The staining can be terminated at that time, but minutes is recommended for proper staining. Q : When protein bands can be clearly detected and background staining does not occur without washing after staining, is the washing procedure still required? A : The washing procedure is not imperative, but a clearer staining can be obtained by washing the gel. When protein bands are light after 60-minute of washing, a clearer staining can be obtained after overnight washing. Q3 : About disposal A3 : does not contain any harmful substances, but is a very dark blue color. Therefore it should be collected in a container prepared for disposal. Q4 : As for the sensitivity of the staining A4 : Some protein bands visible to approx. 0 ng Figure : Stain using ~% gel Sample: Lane,, and : Molecular Weight Marker, High Range (Wako Cat.#34-40); Lane 3,4 and 0 : Molecular Weight Marker, Middle Range (Wako Cat.#3-4) Lane, 6, 7, 8 and 9 : 0,,.,. and 0.6Ug of BSA, respectively ml L RT Related Products Molecular Weight Marker, High Range Molecular Weight Marker, Middle Range ml ml ~ 0 Wako Product Update No.0 Online Catalog :

5 3. Proteome Research Gel Proteomics Add high-purity Matrix CHCA (#037-96) SA (#9-336) DHB (#044-90) See page # Protein identification Analysis of posttranslational protein modification Sampling Electrophoresis In-gel digestion Negative Gel Stain MS Kit Lysyl Endopeptidase, MS (#-006) (#93-770) See page #3 Trypsin, from Porcine Pancreas, Silver Stain MS Kit MS (#0-9) (#99-890) See page #3, 4, See page #4 Mass spectrometry by MALDI-TOFMS a. Silver Stain MS Kit Silver Stain MS Kit Cat. # tests This silver staining kit is produced for mass spectrometric sequencing of protein separated by polyacryalamide gel electrophoresis, based on the method described by Shevchenko et al. By silver staining, protein is rarely modified chemically due to omitting treatment of glutaraldehyde and is detected at sub-nanogram level on the electrophoretic gel. Detectable limit of protein : ng Ideal for MS analysis because rarely modified protein is obtained due to omitting glutaraldehyde treatment. Procedure SDS-PAGE gel Figure. SDS-PAGE analysis using Silver Stain MS Kit Samples were molecular weight markers, containing myosin, B- D-galactosidase, BSA, aldolase, carbonic anhydrase and myoglobin [Kit Contents]. Enhancing Stock Soln. x 00 ml. Staining Stock Soln. x 00 ml 3. Developing Stock Soln. x 00 ml 4. Developing Powder x 0 g. Stopper x 00 ml 6. De-staining Soln. A x 0 ml 7. De-staining Soln. B x 0 ml Silver Stain MS Kit Fixing Soln. Shake 0 min. Fixing Soln. Shake 0 min. Wash 0 min. Enhancing Soln. Shake min. Wash min. x Staining Soln. Shake 0 min. Wash min. x Developing Soln. Shake 3 ~ 0 min. Stopper Shake min. Wash min. x 3 The excised band a b c d e Figure. SDS-PAGE analysis of proteins using Silver stain MS Kit a: rat phosphorylase (97k); b: bovine serum albumin (66k); c: hen egg white ovalbumin (4k); d: bovine carbonic anhydrase (3k); e: soybean trypsin inhibitor (k) (00 ng each) De-staining mixture Leave min. Mass Spectrometry [Reference]. Shevchenko, A., et al., Anal.Chem., 68, 80 (996). Farzin, G., et al., Electrophoresis, 0, 60 (999) Figure 3. MALDI-TOF/MS of rabbit phosphorylase The band was excised and treated with Lysyl Endopeptidase (#-043). Following the in-gel digestion and preparation, the sample was analyzed on MALDI-TOF mass spectrometer. (These data were provided by Dr. Y. Wada at Osaka Medical Center, Japan.) Silver Stain MS Kit 0 tests ~ 0 Wako Product Update No.0 3

6 3. Proteome Research b. Negative Gel Staing MS Kit Negative Gel Staing MS Kit Cat. # It is known that protein bands separated by SDS/polyacrylamide gel electrophoresis (SDS-PAGE) can be visualized as transparent bands against the background of milky white gel stained by negative gel stain containing a Zn/imidazol reagent. We have improved the method using a new imidazol derivative reagent, which allows a clear and stable image of protein bands on the gel as sensitive as that by silver staining, in as little as 0 minutes. The staining technique is useful to obtain the clear and sensitive resolution pattern of the gel before immunoblotting as well as to excise and purify the band of interest from the gel without significant deterioration of amino acid residues for the subsequent studies of protein such as sequencing and mass analysis of peptide. Detectable limit of protein: ng Sensitive and Simple procedure with -step staining in ~ 0 [Kit Contents]. Staining Solution A x 00 ml. Staining Solution B x 00 ml 3. De-staining Solution x 00 ml minutes Optimized the staining intensity by either de-staining the excess stain or re-staining following de-stain of the gel. Applicable to proteome analysis by mass spectrometry and sequencing Procedure Figure. SDS-PAGE analysis of proteins using Negative Gel Stain MS Kit Negative Gel Stain MS Kit SDS-PAGE gel Staining Sol. A Shake ~ 0 min. Wash ~ 0 sec. x 3 Staining Sol. B Shake 0 ~ 60 sec. Wash min. x 3 Mass Spectrometry Check staining intensity of the gel against a sheet of black paper a : rat phosphorylase (97k); b : bovine serum albumin (68k); c : hen egg white ovalbumin (4k); d : bovine carbonic anhydrase (3k). Lane : 0 ng; Lane : 00 ng; Lane 3: 0 ng Figure. MALDI-TOF/MS of rat phosphorylase Figure 3. SDS-PAGE analysis of MW markers using Negative Gel Stain MS Kit run on gel. Figure 4. Negative Gel Stain MS Kit-stained -D gel. Pollen tube protein was applied to a dry strip 3-0. The second dimension was performed on a % gel. The band was excised and treated with trypsin. Following the in-gel digestion and preparation, the sample was analyzed on MALDI-TOF mass spectrometer. (These data were provided by Dr. Y. Wada at Osaka Medical Center, Japan.) [Reference] Fernandez-patron, et al., Anal. Biochem., 4, 63 (99) Negative Gel Stain MS Kit 0 tests Room Temperature 4 Wako Product Update No.0 Online Catalog :

7 3. Proteome Research c. High-purity Matrix for MALDI-TOFMS analysis CHCA, SA and DHB Add High-purity Matrix, such as Wako s CHCA, SA or DHB Protein identification Analysis of posttranslational protein modification Sampling Electrophoresis In-gel digestion Mass spectrometry High purity matrix is mixed with a test sample and used for proteome analysis by mass spectrometry (MALDI-TOFMS). High purity matrix gives good mass spec reading because of its high purity by recrystallization Effects of recrystallization of A-Cyano-4-hydroxycinnamic Acid (CHCA) When comparing with MALDI-TOFMS data for commercial CHCA, it was found that recrystallized CHCA (Wako ) gives clearer mass spec reading with less background noise. (These data were provided by Dr. Wada Y. at Osaka Medical Center, Japan) Untreated CHCA Recrystallized CHCA [Product List] Untreated CHCA with a peptide sample Product A-Cyano-4-hydroxycinnamic Acid [CHCA] Sinapic Acid [SA],-Dihydroxybenzoic Acid [DHB] Recrystallized CHCA with the peptide sample for proteome research x 0 mg x 0 mg x 0 mg ~0c Matrix for MALDI-TOFMS Related Products Product Lysyl Endopeptidase, Mass Spectrometry Trypsin, from Porcine Pancreas, Mass Spectrometry for proteome research x 0 ug x 0 ug -0c Wako Product Update No.0

8 Cell Culture -Free Cell Freezing BAMBANKER TM Cat. # (0 ml) Keep at ~ 0C BAMBANKER TM is a serum-free medium for long-term freezing at -80c and preservation of valuable culture cells such as tumor cells and normal cells.. Ready-to-use medium for preservation of cells. Usable without diluting 3. No program freezer is required 4. Rapid & long-term freezing for preservation in a deep freezer (-80C). -free [Operating Procedure]. Cell Freezing Wako Product Update No. 0 Centrifuge Remove the supernatant Add BAMBANKER TM and suspend cells at x 0 ~ x 0 7 cells/ml -80C Transfer cell suspension from culture vessel to a centrifuging tube Aliquot the suspension into cryotubes. [Comparison of TM with other relevant products] -Cryopreservation Test- <Result> * : period of Cell Line at -80C Media tested. TM (LYMPHOTEC Inc.). with serum (Company A) 3. -free (Company A) P3U (mouse myeloma cell line) Cell number/vial.0 x 0 6 K6 (human leukemia cell line) Cell number/vial 3.0 x 0 6 OKT4 (mouse hybridoma) Cell number/vial.3 x 0 6 Daudi (human B cell line) Cell number/vial 9. x 0 human gastric epithelial cells Cell number/vial.0 x year* 0 year* 0 year* 0 year* 0 0 months* free -free -free -free -free human r st cells Cell number/vial.0 x months* 0 human B cell line Cell number/vial.0 x months* PC (rat-derived adrenal pheochromocytoma) Cell number/vial.0 x 0 6 months* 0 monkey B cell line Cell number/vial.0 x months* free -free -free -free Wako Cat. No BAMBANKER TM 0 ml Keep at ~ 0c BAMBANKER TM is manufactured by (Tokyo, Japan) LYMPHOTEC Inc. BAMBANKER 6 Listed products are intended for laboratory research use only, but not to be used for drug, food or human use. Please visit our online catalog to search for other products from Wako ; This brochure may contain products that cannot be exported to your country due to regulations. Bulk quote requests for some products are welcomed. Please contact us. Wako Pure Chemical Industries, Ltd. -, Doshomachi 3-Chome Chuo-Ku, Osaka , Japan Tel: Fax: Online Cat.: Wako Chemicals USA, Inc. com Head Office: 600 Bellwood Road, Richmond, VA 337 Toll-Free (U.S. only): Tel: / Fax: Los Angeles Sales Office: 6 Alton Parkway, Suite D, Irvine, CA 968 Tel: / Fax: XIBK Wako Chemicals GmbH Nissanstraße, D-4468 Neuss, Germany Tel: Fax: