Enabling Immunoprecipitation with the Rainin PureSpeed System. Rishi Porecha Rainin Instrument LLC

Transcription

1 Enabling Immunoprecipitation with the Rainin PureSpeed System Rishi Porecha Rainin Instrument LLC

2 PureSpeed Tip Product Description Series of tips that have been modified to incorporate a specific volume of purification resin at the distal end of the tip Eight types of resin ProA, ProG, NiNTA, IEX, C18 Variable resin volumes from small to high mass capacity All PureSpeed Tips operate with E4XLS 2

3 Other Components of the PureSpeed System Tips in Clamshells of 12 Setup of PureSpeed System 3

4 Protein Immunoprecipitation Direct and Indirect What is Immunoprecipitation (IP)? A technique to isolate and detect individual proteins in a mixture of thousands of proteins. Protein A and G resins are used to bind an antibody which bind to another protein. This is useful to understand protein function and serves as a basis to understand protein/protein interaction (coip). Capture Wash Elute Elute Indirect Direct IP Antibody Desired (purple) towards Protein the antigen with Other protein Proteins is bound onto ProA or G resin. Wash Introduce away excess Antibody antibody Purify Purify Complex Complex

5 Pictorial Description of Chromatin Immunoprecipitation (ChIP)

6 Loose Agarose Beads Loose Agarose/Sepharose Beads Protein A or G Resin (Various Vendors) Uses centrifugation and pipetting to pellet beads and remove supernatants. Large elution volume dilutes the final IP d sample. Cannot process samples in parallel, only in sequence. Tedious protocol, additional samples add significantly more time.

7 Magnetic Beads Magnetic Bead Systems Protein A or Protein G (Various Vendors) Uses a highpowered magnet and pipetting to pellet beads and remove supernatants. Expensive magnet that has limited functionality. Tedious protocol, additional samples add significantly more time.

8 Ladder Ab Antigen Mix IP using 20/200 µl PureSpeed Protein G Tips 3 Antigen GSThPAK1PBD ProG Resin Bead 1 AntiGST Antibody 2 Heavy Chain Experiment: Compare PureSpeed with the competition in direct and indirect IP. Dynabeads Protein G suggests direct IP, while Pierce Protein G Agarose suggests indirect IP. Light Chain Use 10 µg Antibody, 5 µg antigen, and 125 µg E. coli total protein. Use manufacturer s protocols. Omit antibody, antigen, or both for controls.

9 PureSpeed Functions in Direct and Indirect IP AntiGST Antibody GSTTagged Antigen Protein E. coli Protein Solution Direct IP Indirect IP 97 kda 66 kda 45 kda Ab Heavy Chain Antigen Protein 31 kda Ab Light Chain L S D S I 55 min 30 min

10 PureSpeed Protein G Versus Dynabeads Protein G AntiGST Antibody GSTTagged Antigen Protein E. coli Protein Solution PureSpeed Dynabeads 97 kda 66 kda 45 kda Ab Heavy Chain Antigen Protein 31 kda Ab Light Chain L S P S D Time: 55 min For 6 Samples Time: 69 min For 6 Samples

11 PureSpeed Protein G Versus Pierce Protein G Agarose AntiGST Antibody GSTTagged Antigen Protein E. coli Protein Solution PureSpeed Protein G Agarose 97 kda 66 kda 45 kda Ab Heavy Chain Antigen Protein 31 kda Ab Light Chain L S P S A Time: 30 min For 6 Samples Time: 190 min For 6 Samples

12 PureSpeed ProA Provides LowBackground ChIP Data qpcr qpcr Processed Data Western Blot Input Negative Control H3K27Ac

13 PureSpeed IP in the Literature Peer Reviewed Examples These researchers' ultimate goal is to develop peptide chips as diagnostic screening tools against autoimmune diseases They are developing a technique based on PureSpeed to identify peptides that autoantibodies bind to Their experimental workflow involves immunoprecipitating peptide fragments from proteolyzed protein samples prior to mass spectrometry identification

14 PureSpeed IP in the Literature Peer Reviewed Examples The authors discovered that a lncrna Lethe is transcribed upon cellular treatment with proinflammatory TNFα Lethe serves as a negative feeback regulator of the inflammatory response through NFκB Here, the authors used PureSpeed with Protein A tips to carry out chromatin immunoprecipitation procedures

15 Conclusions PureSpeed can carry out protein immunoprecipitation Chromatin immunoprecipitation is also possible with PureSpeed PureSpeed provides a rapid immunoprecipitation protocol and data advantages Researchers have begun to use the PureSpeed System in peerreviewed studies