SNAP

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SNAP/ CLIP TAGGING :

SNAP/ CLIP TAGGING Presented by : Jisha Shamsudeen Mpharm Part 2 College of Pharmaceutical Sciences, Trivandrum

CONTENTS:

SNAP CLIP TAGGING TECHNIQUE OF TAGGING PURPOSE SUMOYLATION BENEFITS OF TAGGING CONCLUSION REFERENCES CONTENTS

SNAP- CLIP TAGGING:

The SNAP/ CLIP tag labelling technology was developed by Johnsson and Co- workers The tagging technique was derived from human DNA repair protein, O-alkyl guanine dealkyl transferase .(h-AGT). SNAP- CLIP TAGGING

PRINCIPLE OF TAGGING:

The tagging makes use of total internal fluorescence reflection technique. The tagging could be carried out simultaneously in different protein substrates at the molecular level and different protein targets are determined PRINCIPLE OF TAGGING

PURPOSE OF TAGGING:

The tagging technique is employed for the simultaneous measurement of intracellular SUMOylations at molecular level (post translational modifications) and also for single molecule detection . PURPOSE OF TAGGING

TAGGING TECHNIQUE:

TAGGING TECHNIQUE

STAGES IN TAGGING:

Simultaneous SUMOylation Fluorescent labelling TIRF imaging. Single molecule detection.. STAGES IN TAGGING

PROCEDURE FOR TAGGING:

Generate Snap- Clip tagged Sumo proteins The active versions of cDNA of SUMO-1 and SUMO-2 proteins were amplified and inserted into p-SNAP and p-CLIP vector. PROCEDURE FOR TAGGING

PROCEDURE FOR TAGGING:

2 . Generate Snap/Clip –flag protein constructs HEK 293T cells were either transfected with FLAG p53 and RanGAP-1 constructs alone or along with 2.5microgram SNAP SUMO-1 and CLIP Sumo-1 expression constructs using the standard Calcium phosphate precipitation method … PROCEDURE FOR TAGGING

PROCEDURE FOR TAGGING:

Cells were incubated with 5 micrometer H2O2 for 20 minutes or treated with 0.1 micromolar Calyculin A for 45 minutes before lysis PROCEDURE FOR TAGGING

PROCEDURE FOR TAGGING:

3.Sonication and Incubation with affinity resin Cells were sonicated in the lysis buffer and cell debris was removed by centrifugation and finally incubated with substrates BG/BC in the affinity medium at 25 degree celsius for 1 hour PROCEDURE FOR TAGGING

PROCEDURE FOR TAGGING:

After extensive washing the conjugates were eluted from the beads with 0.1 M glycine,and the elution was neutralised with the neutralization buffer… PROCEDURE FOR TAGGING

DETECTION OF TAGGED- FLAP CONSTRUCTS:

DETECTION OF TAGGED- FLAP CONSTRUCTS

DETECTION OF TAGGING SUBSTRATES:

Finally the readings were observed with TIRF spectroscope and the findings were observed with single photon detectors at different wavelengths 647 and 488 nm. DETECTION OF TAGGING SUBSTRATES

SUMOYLATION:

Sumoylation is the covalent attachment of Ubiquitin like protein to various protein targets. More than 750 targets are involved in the process of sumoylation . These targets include tumour suppressors, telomere binding proteins, cell cycle regulators and viral proteins SUMOYLATION

SNAP/ CLIP TAGGING:

SNAP/ CLIP TAGGING

CONCLUSION:

SNAP/ CLIP technique has already been used in the tagging of different proteins like human estrogen receptor, adrenergic b2 receptor and different receptors.. This technique is a non destructive technique as it only encodes the cell surface proteins. Newer tagging techniques like ACP/ MCP are cell lysing tagging technique.. CONCLUSION

REFERENCES:

1.Yang et al, Journal of Angenwandte Chemie,2013, Wiley publishers, pg no : 719-722 2. Textbook of Medicinal Chemistry by Graham Patrick. REFERENCES

THANKYOU:

THANKYOU