Nuclear Extraction Kit
Contents
1. Panomics Nuclear Extraction Kit For Use with Transcription Factor Assays User Manual AY2002 P N 13938 Rev B 091407 Panomics Inc Nuclear Extraction 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 Nuclear Extraction Kit from Panomics If a paper cites a Panomics 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 00200 eee eae Who Should Read this Manual What this Manual Covers 0000 cece eee Safety Warnings and Precautions For More Information 20000000 ee2. Invitrogen P N 14190 144 Rocking platform VWR Rocking Platform Model 100 or equivalent Centrifuge Eppendorf P N 5804R Cell scraper for adherent cell types Costar P N 3010 or equivalent Protein determination kit Bio Rad DC Protein Assay Kit P N 500 0112 or equivalent Microcentrifuge tubes MLS 15 mL conical centrifuge tubes MLS Adjustable single and multi channel precision MLS pipettes Nuclear Extraction Kit User Manual Cell Preparation Cell Preparation Growing Cells In all cases cells are grown to 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 x 108 cells well Nuclear Extraction Procedure for Cultured Cells Assay Guidelines IMPORTANT All components and PBS must be kept on ice at all times Buffer A and B Working Reagents must be kept on ice and should be used within 2 hours of preparation Preparing Working To prepare working reagents Reagents Step Action 1 Prepare 1 mL of Buffer A Working Reagent a Combine 1 mL Buffer A 10 pL DTT b Invert to mix 10 uL Protease Inhibitor 10 uL Phosphotase
3. 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 150 300 pg dish 6 well plate 50 100 ug well Preparing Nuclear To prepare nuclear extract for suspension cells Extracts From Suspension Cells SteP Action 1 Transfer cells to a 1 5 mL or 15 mL centrifuge tube as appropriate 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 Following the second wash step ensure that the 1X PBS solution is completely removed from the cells Note Before the second centrifugation if necessary transfer contents from the 15 mL centrifuge tube to a 1 5 mL microcentrifuge tube Immediately add the appropriate volume of Buffer A Working Reagent to cell pellets Mix by pipetting up and down several times Vessel Buffer A Working Reagent 100 mm culture dish 1 mL dish 6 well plate 250 uL well Transfer tube s to an ice bucket and transfer ice bucket to a rocking platform at 200 rpm for 10 minutes Centrifuge samples at 14 000 x g for 3 minutes at 4 C Discard the supernatant s and keep the pellet s on ice Add 150 uL of Buffer B Working Reagent to each pellet and vortex at highest setting for 10 seconds The pellet w
4. prepare Working Reagents Step Action 1 Prepare 1X Buffer A Buffer B and PBS using nuclease free sterile water Note 1X preparations are good for 6 months at 4 C 2 Prepare Buffer A Working Reagent in a clean sterile tube a Combine the following for each 0 5 grams of tissue 2 5 mL 1X Buffer A 25 uL 100 mM DTT 25 uL Protease Inhibitor 25 uL Phosphatase Inhibitor 25 uL Phosphatase Inhibitor Il b Invert to mix c Keep on ice Nuclear Extraction Kit User Manual Nuclear Extraction Procedure for Whole Tissue To prepare Working Reagents continued Step Action 3 Prepare Buffer B Working Reagent in a clean sterile 1 5 mL tube a Combine the following for each pellet 145 5 uL 1X Buffer B 1 5 uL Protease Inhibitor 1 5 uL Phosphatase Inhibitor 1 5 uL Phosphatase Inhibitor II b Invert to mix c Keep on ice Preparing Nuclear To prepare nuclear extracts from whole tissue Extract From Whole Tissue SteP Action 1 Prepare initial homogenate a Weigh at least 0 5 grams of tissue and dice into very small pieces using a clean razor blade b Place pieces into a pre chilled clean Dounce homogenizer c Homogenize the sample with 15 20 strokes and keep on ice On ice add 1 5 mL of Buffer A Working Reagent per each 0 5 gram of tissue and homogenize with another 15 20 strokes Incubate on ice for 15 minutes Centr
5. Inhibitor 10 uL Phosphotase Inhibitor II Scale preparation of Buffer A Working Reagent based on experimental requirements Use the table below as a guide Quantity of Buffer A Working Vessel Reagent 100 mm culture dish 1 mL dish 6 well plate 250 uL well Nuclear Extraction Kit User Manual Page 7 Nuclear Extraction Procedure for Cultured Cells To prepare working reagents continued Step Action 2 Prepare 1 mL of Buffer B Working Reagent a Combine 1 mL Buffer B 10 uL DTT 10 uL Protease Inhibitor Cocktail 10 uL Phosphotase Inhibitor 10 uL Phosphotase Inhibitor II b Invert to mix Scale preparation of Buffer B Working Reagent based on experimental requirements Use the table below as a guide Quantity of Buffer B Working Vessel Reagent 100 mm culture dish 150 uL dish 6 well plate 150 uL well Preparing Nuclear To prepare nuclear extracts Extracts 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 2 Following the second wash make sure the PBS is completely removed Add the appropriate volume of Buffer A Working Reagent to the wells Vessel Quantity of Buffer A Working Reagent 100 mm cul
6. clear extraction procedure CAUTION All chemicals 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 Nuclear Extraction Kit About the Nuclear The Nuclear Extraction Kit contains reagents and procedures for the preparation of Extraction Kit nuclear extracts for use in our Transcription Factor Assay kits The Nuclear Extraction Kit contains sufficient reagents for the preparation of 80 nuclear extracts from cultured cells grown in 6 well culture plates or 20 nuclear extracts from cultured cells grown in 100 mm culture dishes Kit Contents and The Nuclear Extraction Kit contains the following components Storage Nuclear Extraction Kit User Manual Nuclear Extraction Kit components Component Quantity Storage Buffer A 20 0 mL 20 C Buffer B 12 0 mL 20 C DTT 100 mM 350 uL 20 C Protease Inhibitor 350 uL 20 C Phosphotase Inhibitor 320 uL 20 C Phosphotase Inhibitor II 320 uL 20 C Page 5 Required Materials and Equipment Not Provided Required Materials and Equipment Not Provided Materials and Page 6 Equipment Item Source 1X PBS
7. e Nuclear Extraction Kit 20 0000 0c eee eee eee About the Nuclear Extraction Kit Kit Contents and Storage 2000 cee eee Required Materials and Equipment Not Provided Materials and Equipment 20 0 00a Cell Preparation 0000 cece eee Growing Cells 000 cece eee eee Nuclear Extraction Procedure for Cultured Cells Assay Guidelines 000000 e eee eee Preparing Working Reagents 00 Preparing Nuclear Extracts From Adherent Cells Preparing Nuclear Extracts From Suspension Cells Nuclear Extraction Procedure for Whole Tissue Assay Guideline Ssma erit ina anea 000 aa a A eee About Whole Tissue Extraction 000 Preparing Working Reagents 000 Preparing Nuclear Extract From Whole Tissue Contacting Panomics 00 0c e eee eee Technical Help 0 2 0 ee eee For Additional Services 00 cee eee eae Procarta Transcription Factor Nuclear Extraction 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 Nuclear Extraction Kit from Panomics to prepare nuclear extracts for use in Panomics Procarta TF EMSA TF ELISA or PD Array kits This manual provides the following Kit contents Required materials and equipment Nu
8. ifuge at 850 x g for 10 minutes at 4 C Discard supernatant Note Atthis point most cells have not been lysed On ice add a second 1 5 mL of Buffer A Working Reagent per each 0 5 gram of tissue and homogenize with another 15 20 strokes Incubate on ice for 15 minutes Transfer the homogenate to microcentrifuge tube s and centrifuge at 14 000 x g for 3 minutes at 4 C Remove and save or discard the supernatant cytosolic fraction and place pellet s on ice If desired store the pellet at 80 C and complete the protocol the following day Add 150 uL of Buffer B Working Reagent to each pellet and vortex at the highest setting for 10 seconds The pellet will detach from the microcentrifuge tube wall and may not disperse into a homogenous solution This is normal Do not attempt to disperse the pellet 10 Position the microcentrifuge tubes horizontally in an ice bucket and transfer the ice bucket to a rocking platform at 150 rpm for 2 hours 11 Centrifuge samples at 14 000 x g for 5 minutes at 4 C 12 Measure the protein concentration of each sample using a protein quantitation assay Then prepare 5 uL aliquots for each of the samples Store samples at 80 C or use immediately Nuclear Extraction Kit User Manual Page 11 Contacting Panomics Contacting Panomics Technical Help For technical questions contact our technical support group by telephone at 1 877 726 6642
9. ill detach from the microcentrifuge tube wall and may not disperse into a homogenous solution This is normal Do not attempt to disperse the pellet Position the microcentrifuge tubes horizontally in an ice bucket and transfer ice bucket to a rocking platform at 200 rpm for 2 hours Centrifuge samples at 14 000 x g for 5 minutes at 4 C Nuclear Extraction Kit User Manual Page 9 Nuclear Extraction Procedure for Whole Tissue To prepare nuclear extract for suspension cells continued Step Action 10 Transfer supernatant s to a new microcentrifuge tube This is your nuclear extract 11 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 150 300 pg dish 6 well plate 50 100 ug well Nuclear Extraction Procedure for Whole Tissue Assay Guidelines About Whole Tissue Extraction Preparing Working Reagents Page 10 IMPORTANT All components and 1X PBS must be kept on ice at all times Buffer A and B Working Reagents must be kept on ice and should be used within 2 hours of preparation Each gram of dry tissue should yield approximately 2 x 107 cells for the isolation of nuclei Each kit provides enough solution to prepare 5 grams of tissue To
10. option 3 or by email at techsupport panomics com US and Canada techsupport_europe panomics com Europe or visit our website www panomics com for an updated list of FAQs and product support literature 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 12 Nuclear Extraction Kit User Manual
11. ture dish 1 mL dish 6 well plate 250 pL 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 Release the cells from the bottom of culture vessel a Using a sterile cell scraper remove the cells b Pipet up and down several times to disrupt the cell clumps Avoid creating bubbles c Rinse the bottom of the culture vessel with the cell suspension to maximize the cell yield 6 Transfer each sample to a 1 5 mL microcentrifuge tube and centrifuge at 14 000 x g for 3 minutes at 4 C 7 Remove and discard the supernatant s and keep the pellet s on ice Nuclear Extraction Kit User Manual Nuclear Extraction Procedure for Cultured Cells To prepare nuclear extracts continued Step Action 8 Add 150 uL of Buffer B Working Reagent to each pellet and vortex at highest setting for 10 seconds The pellet will detach from the microcentrifuge tube wall and may not disperse into a homogenous solution This is normal Do not attempt to disperse the pellet Position the microcentrifuge tubes horizontally in an ice bucket and transfer ice bucket to a rocking platform at 200 rpm for 2 hours 10 Centrifuge samples at 14 000 x g for 5 minutes at 4 C 11 Transfer supernatant s to a new microcentrifuge tube This is your nuclear extract 12 Measure the protein concentration of each sample using a protein quantitation assay Then
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