Procarta™ Transcription Factor Whole Cell Lysis Kit
Contents
1. Original Culture Vessel Quantity of Lysis Buffer Il Working Reagent 100 mm culture dish 100 uL dish 6 well plate 25 uL well 24 well plate 10 pL well 96 well plate 5 uL well 6 Transfer tube s or culture plate to an ice bucket and place on a rocking platform at 200 rpm for 1 hour 7 For microcentrifuge tubes pipet up and down several times and centrifuge at maximal speed 12 000 x g for 3 minutes at 4 C Note the orientation of tube in centrifuge as pellets may not be visible For PCR plates pipet up and down several times and centrifuge the PCR plate at 2 250 x g for 5 minutes 8 Transfer supernatant s to a new microcentrifuge tube or a new PCR plate this is your whole cell lysate 9 Measure the protein concentration of each sample using a protein quantitation assay sold separately Then prepare 5 uL aliquots for each of the samples Store samples at 80 C or use immediately in Procarta TF Assay Kit Vessel Typical Protein Yield 100 mm culture dish 300 800 ug dish 6 well plate 200 500 ug well 24 well plate 40 80 ug well 96 well plate 20 40 ug well Contacting Panomics Technical Help For technical questions contact our technical support group by telephone at 1 877 726 6642 option 3 or by email at techsupport panomics com US and Canada or techsupport_europe panomics com Europe or visit our website www panomics com for an updated list of FAQs and product support li2. 1 5 mL or 15 mL centrifuge tube as appropriate and centrifuge at 500 x g for 5 minutes For 96 well plates transfer cells to a PCR plate and centrifuge at 500 x g for 5 minutes Remove the culture media and wash cells by resuspending in 1 mL of cold 1X PBS followed by centrifugation at 500 x g for 5 minutes Repeat wash step For 96 well plates remove the culture media and wash cells by resuspending in 200 uL of cold 1X PBS followed by centrifugation at 500 x g for 5 minutes Repeat wash step Following the second wash step ensure that the 1X PBS solution is completely removed from the cells Note Before the second centrifugation transfer contents from the 15 mL centrifuge tube to a 1 5 mL microcentrifuge tube Immediately add the appropriate volume of Lysis Buffer 1 Working Reagent to cell pellets Mix by pipetting up and down several times Original Culture Vessel Lysis Buffer I Working Reagent 100 mm culture dish 1 mL dish 6 well plate 250 puL well 24 well plate 100 uL well 96 well plate 50 puL well Transfer tube s or culture plate to an ice bucket and place on a rocking platform at 200 rpm for 10 minutes Procarta Transcription Factor Whole Cell Lysis Kit User Manual Page 9 Contacting Panomics To prepare whole cell lysates from suspension cells continued Step Action 5 Add the appropriate volume of Lysis Buffer II to the cells
3. Factor Whole Cell Lysis Assay Kit 2000 5 About the Transcription Factor Whole Cell Lysis Kit 5 5 Kit Contents and Storage 2 eh 5 Required Materials and Equipment Not Provided 002020 eee 6 Materials and Equipment 0 00 cece eee 6 Cell Preparation 3c eru utere ted waite ek aA eg EAS E 7 Growing Gells 5 4 aie Rem equ ted eee taies 7 Whole Cell Lysis Procedure 2 0 0 00 ccc eh 7 Assay Guidelines s serieta rore ewan Me erbe x bet dada petu 7 Preparing Working Reagent 000 e eee eh 7 Preparing Whole Cell Lysates From Adherent Cells 2 8 Preparing Whole Cell Lysates From Suspension Cells 00 9 Contacting PANOMICS i re eR Ba ace EE AEE EORR E REAN 10 Technical Help 3544 dcr Shea eid dob see MeL Leek A Padum 10 For Additional Services oso cera Teo a iney bee eee hr 10 Procarta Transcription Factor Whole Cell Lysis Kit User Manual iii About the User Manual About the User Manual Who Should Read this Manual What this Manual Covers Safety Warnings and Precautions For More Information Anyone that has purchased a Procarta Whole Cell Lysis Kit from Panomics to prepare whole cell lysates for use in Panomics Procarta Transcription Factor TF Assay Kits This manual provides the following Kit contents Required materials and equipment Whole cell lysis procedure CAUTION All chemical
4. dish 100 uL dish 6 well plate 25 uL well 24 well plate 10 pL well 96 well plate 5 uL well 6 Transfer culture vessel s to an ice bucket and transfer ice bucket to a rocking platform at 200 rpm for 1 hour 7 Pipet up and down several times then transfer each sample to a 1 5 mL microcentrifuge tube and centrifuge at 14 000 x g for 3 minutes at 4 C Note the orientation of tube in centrifuge as pellets may not be visible For 96 well plates pipet up and down several times then transfer well contents from the cell culture plate to a PCR plate and centrifuge the PCR plate at 2 250 xg for 5 minutes 8 Transfer supernatant s to new microcentrifuge tubes or a new PCR plate this is your whole cell lysate Procarta Transcription Factor Whole Cell Lysis Kit User Manual Whole Cell Lysis Procedure To prepare working reagent continued Step Action 9 Measure the protein concentration of each sample using a protein quantitation assay sold separately Then prepare 5 uL aliquots for each of the samples Store samples at 80 C or use immediately in Procarta TF Assay Kit Vessel Typical Protein Yields 100 mm culture dish 300 800 ug dish 6 well plate 200 500 ug well 24 well plate 40 80 ug well 96 well plate 20 40 ug well Preparing Whole To prepare whole cell lysates from suspension cells Cell Lysates From Suspension Cells 9f P Action 1 Transfer cells to a
5. Procarta Transcription Factor Whole Cell Lysis Kit User Manual amp h Panomics Panomics Inc Procarta Transcription Factor Whole Cell Lysis Kit User Manual Copyright Copyright 2006 Panomics Inc All rights reserved Trademarks Procarta is a trademark of Panomics Inc Citing Procarta in Publications When describing a procedure for publication using this product we would appreciate it if you would refer to it as the Procarta TF Whole Cell Lysis Kit If a paper cites a Procarta product and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at techsupport panomics com or via fax at 510 818 2610 Disclaimer Panomics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Panomics Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Contents About the User Manual 0 0 00 cee 5 Who Should Read this Manual lisse 5 What this Manual Covers 0 0000 cece ses 5 Safety Warnings and Precautions 0 0 0 cece eee 5 For More Information liiis ena 5 Procarta Transcription
6. is Buffer 1 10 pL DTT 10 uL Protease Inhibitor Cocktail Scale Lysis Buffer preparation according to experimental design Use the table below as a guide Quantity of Lysis Working Vessel Reagent 100 mm culture dish 1 mL dish 6 well plate 250 uL well 24 well plate 100 uL well 96 well plate 50 uL well Procarta Transcription Factor Whole Cell Lysis Kit User Manual Page 7 Whole Cell Lysis Procedure Preparing Whole To prepare working reagent Cell Lysates From Adherent Cells Page 8 Step Action 1 Remove the culture media from all wells and wash cells twice with an appropriate volume of cold 1X PBS Vessel Quantity of PBS 100 mm culture dish 10 mL dish 6 well plate 1 mL well 24 well plate 500 uL well 96 well plate 200 uL well 2 Following the second wash make sure the PBS is completely removed Add the appropriate volume of Lysis Buffer Working Reagent to the wells Vessel Quantity of Lysis Buffer I Working Reagent 100 mm culture dish 1 mL dish 6 well plate 250 uL well 24 well plate 100 uL well 96 well plate 50 puL well 4 Transfer culture vessel s to an ice bucket and transfer ice bucket to a rocking platform at 200 rpm for 10 minutes 5 Add the appropriate volume of Lysis Buffer II Working Reagent to the wells Vessel Quantity of Lysis Buffer Il Working Reagent 100 mm culture
7. s should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice Note This product is intended for research use only For information about the Procarta products mentioned in this manual visit our website at www panomics com Procarta Transcription Factor Whole Cell Lysis Assay Kit About the Transcription Factor Whole Cell Lysis Kit Kit Contents and Storage The Procarta TF Whole Cell Lysis Kit contains reagents and procedures for the preparation of whole cell lysates for use in our Procarta TF Assay Kits The Procarta TF Whole Cell Lysis Kit contains sufficient reagents for the preparation of 40 whole cell lysates from cultured cells grown in 6 well culture plates or 240 whole cell lysates from cultured cells grown in 96 well culture plates The Procarta Transcription Factor Whole Cell Lysis Assay Kit contains the following components Procarta TF Whole Cell Lysis Kit components Component Quantity Storage Lysis Buffer 12 0 mL 20 C Lysis Buffer II 1 2 mL 20 C DTT 120 uL 20 C Protease inhibitor cocktail 120 uL 20 C Procarta Transcription Factor Whole Cell Lysis Kit User Manual Page 5 Required Materials and Equipment Not Provided Required Materials and Equipment Not Provided Materials and Equi t I
8. tem Source uipmen quip 1X PBS Invitrogen P N 14190 144 Rocking platform VWR Rocking Platform Model 100 or equivalent Centrifuge Eppendorf 5804R Protein determination kit Bio Rad DC Protein Assay Kit P N 500 01 12 or equivalent Microcentrifuge tubes Major laboratory supplier MLS 15 mL conical centrifuge tubes MLS Adjustable single and multi channel precision pipettes MLS PCR plates MLS Page 6 Procarta Transcription Factor Whole Cell Lysis Kit User Manual Cell Preparation Cell Preparation Growing Cells In all cases cells are grown to about 90 confluence The following table provides recommendations for the cell requirements for each culture vessel type However it is important to realize that cell types vary in size and actual numbers of cells vessel may vary Use the table below as a guide Culture Vessel Cell Number 100 mm culture dish 1 x 107 cells dish 6 well plate 1 5 x 106 cells well 24 well plate 2 5 x 105 cells well 96 well plate 2 5 x 104 cells well Whole Cell Lysis Procedure Assay Guidelines IMPORTANT All components and PBS must be kept on ice at all times Lysis Buffer 1 Working Reagent must be kept on ice and should be used within 2 hours of preparation Preparing Working To prepare working reagent Reagent Step Action 1 For preparation of 1 mL Lysis Buffer 1 Working Reagent combine the following reagents then invert tube to mix 1 mL Lys
9. terature For Additional For information about Panomics products or for ordering information contact your Services Regional Sales Manager or visit our website at www panomics com Page 10 Procarta Transcription Factor Whole Cell Lysis Kit User Manual
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