Brc H700 Manual

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What is the BRC Series? This camera has the best sensitivity of the BRC Series; it therefore delivers superior performance in dimly lit environments, such as concert or wedding halls. Users can choose either HD or SD system components.Normally set to the VISCA RS-232C IN connector of the next 0. With this setting, addresses are assigned to camera in the daisy chain. With this setting, addresses are assigned to the cameras automatically in the connected order by pressing the POWER button while holding down the RESET button on the RM-BR300 Remote Control Unit. No audio signal is output from the card. For the the same camera number, they are operated BRC-H700 and BRC-Z700, preset positions from 7 simultaneously with the same IR Remote Com- mander Unit. If you are installing cameras close to 16 are not available. This connection is one example using the RS-422 connection.For the BRC-H700, the BRC-Z700, and the BRC-Z330, preset positions from 7 to 16 are not available. Move the positioner for pan (which moves the camera shooting direction horizontally) control and tilt (which moves the camera shooting direction vertically) control. Both of these cameras adjust the saturation of six colors inde- pendently, and the BRC-H700 is able to modulate six colors simultaneously.For example, if the switch of the BRU- H700 is ON and the switch of the camera is OFF, the menu will be displayed on the video images. DATA MIX switch on the BRC-H700 (rear) DATA MIX switch on the BRC-Z330 (rear) Using another screw may to the ceiling bracket (B), and install the bracket disable the function of the wire rope.To remove the camera For attaching the camera to the ceiling bracket, use. Use four into the spaces prepared in the ceiling bracket screws (not supplied) appropriate for the (B) with the a hole in the front of the ceiling materials of the shelf, etc. Take the proper steps to ensure that the load of the Note cables connected does not cause problems.
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To remove the camera Remove the three screws used to attach the camera in step 7 of “Installation on a shelf, etc. For the BRC-H700 and BRC-Z700, preset positions from 7 to 16 are not available. Move the positioner for pan (which moves the camera shooting direction horizontally) control and tilt (which moves the camera shooting direction vertically) control. This equipment generates, The model and serial numbers are located on the bottom.They do not indicate malfunctions. Operating or storage location White flecks Operating or storing the camera in the following Although the CCD image sensors are produced with locations may cause damage to the camera: high-precision technologies, fine white flecks may be. Extremely hot or cold placeEven when you shoot a subject using maximum Features magnification, clear images with minimum aberration can be obtained by a lens system that consists of 16 lenses of 11 groups containing three aspherical lenses. Compact, HD 3CCD vidThis section introduces these optional products as well as the Tally lamp accessories supplied wSupplied accessories: AC power adaptor (This is a 2-core multi-mode optical fiber cable of 100 m HFBK-TS1 HDV Interface Board (328 feet) long. Supplied accessories: extension plug HFBK-SD1 SD Interface Board Insert the board into the camera or the HD Optical Multiplex Unit to allow conversion of the video signal froThis section describes nine typical system examples with the required components and the main usage of each system. Operating a BRC-H700 Camera Using the Supplied Remote Commander This system allows you: To operate the camera readily from a short distance System configuration BRC-H700 HD video monitor, VTR, etc. Video signal Remote Commander (supplied) STo operate up to seven cameras remotely using a single Remote Control Unit.To operate the camera remotely from a distance up to 1,000 m (3,281 feet).
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To transmit the video signal and control signal of the camera to a distant place using the Optical Fiber Cable System configuration HD video monitor, VTR, etc. BRC-H700 CCFC-M100HG Optical Fiber Cable BRU-H700 Optical Multiplex Unit BRBK-H700 HD Optical Multiplex CTo operate up to seven cameras remotely from a distance up to 1,000 m (3,281 feet). To transmit the video signal and control signal of the cameras to a distant place using the Optical Fiber Cable System configuration BRC-H700 HD video monitor, VTR, etc. BRU-H700 Optical CCFC-M100HG Multiplex Unit Optical Fiber Cable BRBK-H700 HD To control the BRC-H700 cameras remotely from a distance up to 1,000 m (3,281 feet) by transmitting the video signal and control sigTo operate the camera remotely from a distance up to 1,000m (3,281 feet). To transmit the video signal and control signal of the camera, and the audio signal input to the BRBK-H700 Optical Multiplex Card to a distant place using the Optical Fiber Cable System configuration Speakers HD video monitor Microphone BRC-H700 Microphone amplifier AudTo input the video signal of the camera to the HDV video equipment. To input the video signal of the camera and the audio signal via the HFBK-TS1 HDV Interface Board simultaneously to the HDV video equipment.To operate the camera remotely from a distance up to 1,000m (3,281 feet). To transmit the video signal and control signal of the camera, and the audio signal input to the BRBK-H700 Optical Multiplex Card to a distant place using the Optical Fiber Cable. To output the audio sFor details, see “Using the ND Filter” on page 31. Front Rear 1 2 3 With the cable cover attached 890qaqs 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 OFF ON OFF ON 1 2 3 1 2 3 75 75 VISCA RS-422 DC IN 12V VISCA RS-42HFBK-SD1, HFBK-XG1, HFBK-TS1 or BRBK- For details, see “Setting of the BOTTOM switches” H700. on page 19. The slot cover is attached to the camera at the factory. Setting of the BOTTOM switchesPress it again to turn off the menu.
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If you press the button when a lower-level menu is selected, the display goes back to a higher-level menu. DATA SCREEN BACK LIGHT 7 3 STD REV D PAN-TILT buttons 12 3 Press the arrow buttons to perform panning and 45 6 8 P. This camera is ideal for low-light environments. It is also quite common that customers throw outUser manuals are alsoTry Google Search! Venus Optics KX-800 Flexible Macro Twin Flash VEKX800 - Venus Optics KX-800 Flexible Macro Twin Flash VEKX800 Posted by: J Gavin 2020-08-12 05:54:49 I am trying to find a user manual for the Venus Optics KX-800 Flexible Macro Twin Flash VEKX800. Did you miss your activation email ? I picked up two on ebay for 1p, in a state of disarray. It would appear someone has dismantled the tilt mechanisms on both of them and both are missing parts, but some of which came in a plastic bag full of washers, cogs and bearings etc. The goal is to get one working, but I don't know if i have enough bits, or what the under lying faults were. Sony still sell parts for them, but it looks like many of the custom parts are no longer available. Still one of these cameras with just pan working would be an interesting project. Sony BRC-H700 Command List - DISCONTINUED PRODUCT (PDF) 133k; Share the Sony BRC-H700 PTZ Camera. BRC300, 1 manual. BRCH700, 1 do sluchania dcr dvd101 owners manual Model: DCR-DVD 101. Sony Handycam DCR-SX43 Free download of Sony BRCH700 User Manual.Reload to refresh your session. Reload to refresh your session. Wire Flat-head screwdriver 12 34 5678 9 12 3 4 56 78 9 Sony Corporation Printed in Japan If you are here, it was rather the case. However, you are not the only person having problems with keeping the operation manual of all household devices. Below are few guidelines regarding how and why you should collect the product manuals. However, we believe that the manuals should include the most important and needed information about Sony BRC-H700, not to discourage the user to read.
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Obviously, if a device Sony BRC-H700 has multiple advanced functions, we will not avoid a high amount of information in this document. Then, it will be much easier to find it than look through the purchase boxes which have already been thrown away by you or any other of household members. It will be enough to clear the drawer once in a year and throw away any manuals of the devices you do not use anymore. Thus, you can avoid storing any unnecessary documents and keep only those which are valid. You can also download and print the manual Sony BRC-H700 to place it in your drawer. The BRU-H700 HD optical mutliplex unit with the BRBK-H700 HD optical multiplex card enables the BRC-H700 to transmit uncompressed digital data without deterioration through the CCFC-M100HG HD optical fiber cable. The same RM-BR300 remote control unit for the BRC-300 can be used with BRC-H700; it will simultaneously control up to 7 cameras from a distance. Multiplex Unit. Operating Instructions. Mode d’emploi. Manual de instrucciones. GebrauchsanweisungThe model and serial numbers are located on the top. Record the serial number in the space provided below. Refer to these numbers whenever you call upon your. Sony dealer regarding this product. Model No. BRU-H700. Serial No.To avoid electrical shock, do not open thePlease ensure that the socket outlet is installed near theIn the event of abnormal operations, disconnect theLaser Notice. This product contains an optical fiber connecter usingThis product is classified as a CLASS 1 LASERWavelengthOtherwise, your eyes may beFor customers in the U.S.A. This device complies with part 15 of the FCC Rules. Operation is subject to the following two conditions:Part 15 of the FCC Rules. These limits are designed toThis equipment generates,You are cautioned that any changes or modifications notAll interface cables used to connect peripherals must beFor the customers in Canada. This Class A digital apparatus complies with CanadianFor the customers in Europe.
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Warning. This is a Class A product. In a domestic environment,In the case that interference should occur, consult yourTable of Contents. Features. 4. Connecting Cables. 4. Example of System Configuration. 4. Location and Function of Parts. 5. Attaching an Interface Board. 6. Precautions. 7. Specifications. 7. Dimensions. 8. Pin Assignments. 9. Using the VISCA RS-422 Connector Plug. 10Features. The BRU-H700 HD Optical Multiplex Unit is designedCamera. HD cameraThe BRU-H700 is equipped with a camera connector toYou can connect the BRC-H700 HD camera from up toHD Optical Multiplex Card into the HD camera andTwo interface card slots equippedIn addition toXGA and WXGA) and HDV standard signal outputs.Optical connector. BRBK-H700 HD Optical. Multiplex CardRS-232C cableRS-232C cableUse the following connecting cable to connect devicesPart No.HD monitor, etc. CableUnit (this unit)The BRU-H700 allows output of stereo audio lineOptical Multiplex Card inserted into the BRC-H700 HDConnecting Cables. AC power cordCCFC-M100HG Optical. Fiber CableVISCA RS-422 connectors. Note. To start the system, first turn on the power of the BRCH700 camera, then turn on the power of the BRU-H700Location and FunctionOptical Multiplex Card inserted into the camera viaConnect the supplied AC power cord. G CAMERA connector. Connect to the optical connector of the BRBKH700 HD Optical Multiplex Card installed in the. BRC-H700 camera using the CCFC-M100HG. Optical Fiber Cable. A dustproof cap is attached at the factory. FrontAccepts external video sync signals. A Power switchB Power indicator. Lit in green: This unit is in normal operation. Lit in red: The power of the camera connected toFlashing in red: Abnormal operation of this unit. Display the composite video signal on the monitorCheck also theC DATA MIX switch. Set the switch to ON to overlap the menu with theRearSupplies external video sync signals input from the. EXT SYNC IN connector.RGB signal. K VISCA RS-232C IN connector.
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Connect to the RM-BR300 Remote Control UnitL VISCA RS-232C OUT connector. When you connect multiple cameras, connect it toM VISCA RS-422 connector. Connect to the VISCA RS-422 connector of theMultiplex Unit. For the connection to the VISCA RS-422Connector Plug” on page 10.D Card slot. Insert an optional interface board, HFBK-SD1. HFBK-HD1, HFBK-XG1 or HFBK-TS1. The slot cover is attached to the unit at the factory. Loop through output of the audio line signal inputN VISCA FUNCTION switches. These switches are used for the VISCASet to ON for RS-422, or OFF for RS-232C. Switch 2 (Communication baud rateSet to ON for 38400bps, or OFF for 9600bps. Location and Function of PartsSet the address of the camera.CameraBoard. Attach an interface board such as BRBK-301 or BRBK302 (not supplied) to the card slot on the rear of this unit.Align both ends of the interface board with theSwitch 4 OFF OFF ON ON OFF OFF ON ON. Switch 5 OFF OFF OFF OFF ON ON ON ON. Set to ON for output of 50i signal format, or OFFThe camera connected to this unit operates in theNote. Set the switches before you turn on the power of the unit. Interface board (not supplied)To remove the interface board. Loosen the two screws on the interface board and pullPrecautions. Specifications. Operating or storage location. System. Operating or storing the unit in the following locationsVideo signal. Ventilation. To prevent heat buildup, do not block air circulationTransportation. When transporting the unit, repack it as originallyCleaningSynchronization. Aspect ratioCamera input. Video output. LC Duplex Fiber connector (1). D-sub 15-pin. RGB: 0.7 Vp-p (no sync, at 75ohm termination)Component:EXT SYNC IN: BNC type (1). SD sync 2.0 V max. (withEXT SYNC OUT: BNC type (1)VISCA RS-232C OUT: Mini. DIN 8-pin type (1). VISCA RS-422: 9-pin type (1). Control signal format. Data: 8 bit. Stop bit: 1. Audio line output. Maximum output level: 2.8. Vrms. Output impedance 1 kilohms orGeneral. Current consumption 0.4 to 0.3 ATop.
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Power consumption 10 W max. (without optionalOperating temperature. DimensionsMass. Approx. 2.4 kg (5 lb 5 oz). Supplied accessories. AC power cord (1). RS-232C connecting cable (1). RS-422 connector plug (1). Operating Instructions (1). Design and specifications are subject to change withoutFront. Side. Unit: mm (inches)Pin Assignments. VISCA RS-422 connector (connector plug,VISCA RS-232C IN connector (mini DIN 8-pin,No Connection. Pin No.No Connection. Pin. No. Function. At YPbPr. At RGB. COMPONENT COMPONENT setting (atPr-OUTPb-OUTTri-levelPlugFlat-head screwdriver. WireVISCA RS-422 connector.Grasp both ends of the VISCA RS-422 connector plugRS-422 connection is approximately 1,200 m (3,937Avis sur le laser:Longueur d’ondeCeci serait dangereux pour lesPour les utilisateurs au CanadaPour les utilisateurs en Europe. Avertissement. Il s’agit d’un produit Classe A. Dans un environnementDimensions. 8. Brochage. 9. Utilisation de la fiche de connecteurDeux emplacements de carte d’interface. En plus de ceConnecteur optique. Carte multiplex optiqueBRU-H700 (cetLe BRU-H700 permet la sortie des signaux de ligneMoniteur HD, etc. NombreRemarque. BRC-H700 sous tension, puis mettez le module BRUH700 (cet appareil) sous tension. Autrement, le messageEmplacement et. Branchez-y le cordon d’alimentation fourni. Face avantRaccordez-le au connecteur optique de la carteA Interrupteur d’alimentationMettez-la sousClignote en rouge: Fonctionnement anormal de ce. C Commutateur DATA MIX. Placez le commutateur sur ON pour recouvrir le. Placez-le sur OFF pour ne pasI Connecteur EXT SYNC OUT. J ConnecteurLorsque vous raccordezL Connecteur VISCA RS-232C OUTM Connecteur VISCA RS-422. Raccordez-le au connecteur VISCA RS-422 de laD Emplacement de carte.
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N Commutateurs VISCA FUNCTIONFixez une carte d’interface BRBK-301 ou BRBK-302Vous pouvez attribuer manuellement les adressesAdresseCommu- OFF OFF ON ON OFF OFF ON ONCommu- OFF OFF OFF OFF ON ON ON ONPlacez-le sur ON pour la sortie en format de signalCarte d’interface (non fournie)RemarquePour retirer la carte d’interface. Desserrez les deux vis de la carte d’interface et tirez laEmplacement d’utilisation ou deL’utilisation ou le stockage du module dans les endroitsTransport. Pour transporter le module, remballez-le dans son. Nettoyage. Pour enlever les taches tenaces. Synchronisation. Rapport de formatSynchronisation trois niveaux:EXT SYNC IN: type BNC (1)Synchronisation SD 2,0 V max.EXT SYNC OUT: type BNC (1). VISCA RS-232C IN: Type miniDIN 8 broches (1). VISCA RS-232C OUT: TypeFormat du signal de commande. Niveau de sortie maximal:Consommation de courant. ConsommationDimensionsPoidsDimensions. Accessoires fournis. Cordon d’alimentation (1). Fiche de connecteur RS-422 (1). Mode d’emploi (1). Face avant. Appareil: mm (pouces)BrochagePas de connexionPas de connexion. FonctionAvec le. YPbPrAvec leRGB (surPr-OUTPb-OUTFilVISCA RS-422, la connexion du VISCA RS-232CEn caso de operaciones anormales, desconecte la clavija. Longitud de ondaPrecauciones. 7. Especificaciones. 8. Dimensiones. 9. Asignaciones de clavillas. 9Cable de. CA (suministrado). A una tomaMonitor HD, etc. Cable RS-232CCable. Cable RS-232CUnidad de mando a distanciaNotaParte delanteraC Interruptor DATA MIX. Ajuste el interruptor en ON para superponer elParte traseraD Ranura de la tarjeta. Inserte una tarjeta de interfaz opcional, HFBKSD1, HFBK-HD1, HFBK-XG1 o HFBK-TS1.G Conector CAMERAH Conector EXT SYNC IN. I Conector EXT SYNC OUT. J Conector. YPbPr o RGB. K Conector VISCA RS-232C IN. Se conecta a la unidad de mando a distancia RMBR300 (no suministrada). Cuando conecteL Conector VISCA RS-232C OUT. M Conector VISCA RS-422. N Interruptores VISCA FUNCTION.
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Estos interruptores se utilizan para el ajuste de laNormalmente ajustada en “0”. Con esta. RM-BR300 (no suministrado).Interrup- OFF OFF ON ON OFF OFF ON ONInterrup- OFF OFF OFF OFF ON ON ON ONNota. Ajuste los interruptores antes de encender laInserte una tarjeta de interfaz, tal como una BRBK-301 oPrecauciones. Lugar de funcionamiento oAlinee ambos extremos de la tarjeta de interfaz conTransporte. Cuando transporte la unidad, vuelva a empaquetarlaLimpieza. Tarjeta de interfaz (no suministrada)Para eliminar lasPar extraer la tarjeta de interfaz. Afloje los dos tornillos de la tarjeta de interfaz y tire deImpedancia de salida,Especificaciones. SistemaD-sub de 15 clavillas. RGB: 0,7 Vp-p (sinComponente:EXT SYNC IN: Tipo BNC (1). EXT SYNC OUT: Tipo BNC (1). VISCA RS-232C IN: Tipo DINVISCA RS-232C OUT: Tipo. DIN mini de 8 clavillas (1). VISCA RS-422: Tipo de 9Datos: 8 bits. Bit de parada: 1Generalidades. Consumo de corrienteConsumo de potenciaTemperatura de funcionamiento. Temperatura de almacenamiento. DimensionesPeso. Aprox. 2,4 kg (5 lb 5 oz). Accesorios suministrados. Manual de instrucciones (1)Asignaciones de clavillas. Superior. Conector VISCA RS-232C IN (mini DIN de 8DimensionesLateralEspecificacionesVISCA RS-422, y apriete el tornillo de ese cableCableEn ajuste. En ajusteRGB (en. YPbPr. YPbPrEn ajuste. RGB (enPr-OUTPb-OUTVD-OUT deVISCA RS-422 es aproximadamente de 1.200 mUm einen elektrischen Schlag zu. Wartungsarbeiten stets nurBitte stellen Sie sicher, dass eine Netzsteckdose in der. Ziehen Sie im Falle eines anormalen Betriebs den. Netzstecker ab. Laser-Hinweis. Laser, welcher der Norm EN 60825-1 entspricht. Dieses Produkt ist als LASER-PRODUKT KLASSE 1Laserdioden-Information (Lichtleiteranschluss)WarnungDiese Einrichtung kann im. Fall kann vom Betreiber verlangt werden, angemesseneMerkmale. 4. Verbindungskabel. 4. Beispiel der Systemkonfiguration. 4. Lage und Funktion der Teile. 5. Einsetzen einer Schnittstellenkarte. 6. Spezifikationen. 7. Abmessungen. 8.

Stiftbelegung. 9. Verwendung des VISCA RS-422Anschlusssteckers. 10MerkmaleHD-KameraDie BRU-H700 ist mit einem KameraanschlussKomponenten-Videoausgang (D-Sub, 15-polig)HD-SDI-Signale, Signale des VESA-Standards (VGA. XGA und WXGA) und Signale des HDV-Standards,Netzsteckdose. Optische Buchse. Optische HD-MultiplexkarteNetzsteckdoseVideosignal durch Eingabe eines Sync-Signals in dieAusstattung mit Audio-Line-Ausgangsbuchsen. Die Einheit BRU-H700 gestattet die Ausgabe von. NetzkabelLichtleiterkabelEinheit)Komponenteneingang. RS-232C-KabelHD-Monitor usw.Verwenden Sie das folgende Verbindungskabel zum. Kabel. Teile-Nr. Anzahl. RS-232C-KabelNetzsteckdose. Fernbedienungseinheit RM-BR300. Hinweis. RS-232C-KabelUm das System hochzufahren, schalten Sie zuerst die. Kamera BRC-H700 und dann die Einheit BRU-H700Anderenfalls kann dieLage und Funktion der. Teile. Vorderseite. Die Steckplatzabdeckung wurde werksseitig an der. Einheit angebracht.A Netzschalter. Schalten Sie die Kamera BRC-H700 ein, bevor SieB BetriebslampeSie die Kamera ein. Bringen Sie das FBAS-Video-Signal auf dem. Anschluss. C Schalter DATA MIX. Stellen Sie den Schalter auf ON, um das von derStellen SieLichtleiterkabel CCFC-M100HG mit der optischen. Buchse der in die Kamera BRC-H700 eingesetzten. Optischen HD-Multiplexkarte BRBK-H700. Eine Staubschutzkappe wurde werksseitigH Buchse EXT SYNC IN. Dient zur Eingabe externer Video-Sync-Signale. I Buchse EXT SYNC OUT. Liefert in die Buchse EXT SYNC IN eingegebeneJ Buchse. Gibt die Kamerabilder als YPbPr- oder RGBSignale aus. K Buchse VISCA RS-232C IN. Wird mit der Fernbedienungseinheit RM-BR300Wenn Sie mehrereL Buchse VISCA RS-232C OUT. Sie diese Buchse mit der Buchse VISCA RS-232C. M Buchse VISCA RS-422. Verbinden Sie diese Buchse mit der Buchse VISCA. RS-422 der Kamera oder einer anderen Optischen. HD-Multiplexeinheit BRU-H700.D Kartensteckplatz.

Hier wird die optionale Schnittstellenkarte HFBKSD1, HFBK-HD1, HFBK-XG1 oder HFBK-TS1RS-422-Anschlusssteckers“ auf Seite 10. N Schalter VISCA FUNCTION. Lage und Funktion der TeileDiese Schalter dienen zur Einstellung der. Kameraadresse. Einstellung werden die Adressen den KamerasFernbedienungseinheit RM-BR300 (nichtKameraadresseSchnittstellenkarte. Setzen Sie eine Schnittstellenkarte, wie z.B. BRBK-301Richten Sie die Seitenkanten der. Steckplatz aus, und schieben Sie dann die. Schnittstellenkarte einwandfrei bis zum AnschlagDie an diese Einheit angeschlossene KameraSchnittstellenkarte (nicht mitgeliefert)Schnittstellenkarte an. HinweisSo entfernen Sie die Schnittstellenkarte. Schnittstellenkarte, und ziehen Sie die Karte gerade undSpezifikationen. Betriebs- oder Lagerungsort. SystemFernsehsenderVideosignal. Transport. Sie es wieder in der Original-Werksverpackung oder in. Reinigung. Entfernen SieAutomatische Umschaltung.

With respect to antigens, chemically synthesized peptides are well suited for FIXAA because they can be prepared under RNase-free conditions. Another limitation of FIXAA is the difficulty in amplifying full-length V genes from PFA-fixed cells because the PFA-fixation process results in cross-linkage between RNA and proteins, making RNA more recalcitrant to extraction 27. PFA also causes the covalent modification of RNA by mono-methylol, which impairs the reverse transcriptase reaction 28. Although several attempts have been made to address these problems, successful PCR amplification has been limited to short targets 29, 30, 31, 32. We envisage using two ASPC isolation methods, ERIAA and FIXAA, for the generation of mAbs: ERIAA for the isolation of relatively abundant ASPCs with roughly purified antigens; FIXAA for the isolation of rare ASPCs with RNase-free antigens. Taken together, this novel method provides a significant advantage over conventional animal immunization techniques and is particularly well suited for generating mAbs against a variety of phosphorylated proteins using chemically synthesized peptides. Materials and Methods Materials Paraformaldehyde, Phosphatase Inhibitor Cocktail 3, NaF, sodium pyrophosphate, Na 3 VO 4 and other chemicals and reagents were purchased from Sigma-Aldrich ( ). Restriction enzymes and lambda protein phosphatase were purchased from New England BioLabs ( ). Blocking One-P and Chemi-Lumi One Super were purchased from Nacalai Tesque ( ). The sequences of the peptides used in this work are listed in Table 1. OKT10 hybridoma and Jurkat cells were purchased from ATCC. CD38 was labeled with DyLight Fluor Labeling Reagents and purified using Bio-Gel columns (BIO-RAD, ). DyLight-labeled goat secondary antibodies against IgG were purchased from Abcam ( ). Rabbit polyclonal anti-pan-p53 antibody (SC-6243) was purchased from Santa Cruz Biotechnology Inc ( ).

DsRed protein was produced in Escherichia coli BL21 and purified using Superflow Ni-NTA columns (Qiagen, ), as described previously 12. Female Hartley guinea pigs were purchased from Japan SLC, Inc. ( ). Hela cells, Saos-2 cells and cDNAs encoding p53 were provided by the RIKEN BRC ( ) through the National Bio-Resource Project of the MEXT, Japan 33. Table 1 Peptides used in this study. OKT10 hybridomas and Saos-2 cells were grown in RPMI medium containing 10 FBS. Lymph node cell preparation All animal studies were approved by the Committee for Laboratory Animal Care and Use at University of Toyama and the experiments were carried out in accordance with the approved guidelines. At 1 week after the final immunization, the iliac lymph nodes were surgically removed and used for the isolation of ASPCs 12. Isolation of ASPC by FIXAA To inactivate RNases, the FACS sample lines were rinsed with 0.1 sodium hypochlorite followed by RNase-free water. Prior to use, DyLight-conjugated secondary antibodies were diluted (1:250) in PBS-0.1 TritonX100 (PBST) containing 0.01 diethylpyrocarbonate (DEPC). Single-cell sorting and data analysis were performed using a JSAN flow cytometer equipped with an automatic cell deposition unit ( ). The channels used were as follows: anti-IgG in the FL-1 or FL-5 channel, phosphorylated peptide in the FL-1 channel, unmodified peptide or DsRed in the FL-2 channel and DAPI or ER-tracker in the FL-7 channel. Molecular cloning of V H and V L genes from single cells and recombinant antibody expression V H and V L genes were amplified from single cells as previously described, with some modifications 11. The primers used were essentially the same as described before. The PCR-amplified V gene fragments were joined to their respective DNA cassettes to build linear immunoglobulin heavy and light chain genes by TS-jPCR, as described previously 13.

The PCR-amplified V gene fragments were subcloned into a mammalian expression vector by TS-homologous recombination 34. The cell culture supernatant was assayed by ELISA for antigen-binding activity at 3 days after transfection. After washing the plate with PBS, the bound mAbs were detected with HRP-conjugated secondary antibodies. Plasmid construction p53 variants with residues replaced by alanine at positions S15, T18 and S20 were generated by QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). Sequencing (ABI 3730 XL Sequencer) was carried out to confirm all mutations. The plasmids were introduced into Saos-2 cells using CUY21 SC Square Wave Electroporator (Nepa Gene ). Microscopy images with the same exposure settings were taken of immunostained cells with and without phosphatase treatment. The images were captured using an Olympus IX71 fluorescence microscope equipped with a SPOT RT3 digital microscopy camera (SPOT Imaging Solutions, ) and analyzed using 2D Deconvolution MetaMorph software (Molecular Devices, ) or a Leica TCS SP8 Confocal Laser Scanning Microscope ( ). Western blots were imaged using Odyssey Infrared Imaging System (LI-COR Biosciences). All antibody solutions were subsequently used for immunofluorescent analysis and Western blotting as described above. Binding data at different antibody concentrations were globally fit to a simple 1:1 interaction model.We also thank Y Nohara, S Fuji and C Matsui for technical support. Ethics declarations To view a copy of this license, visit Download citation Received: 06 January 2016 Accepted: 11 April 2016 Published: 29 April 2016 DOI: If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Its influence, however, was much greater than that—manufacturers around the world began building jeeps and similar designs, either under license or not—at first primarily for military purposes, but later also for the civilian market.