The gene encoding the full-length Ovalbumin protein was ordered as a synthetic gene fragment (IDT). BP Clonase II (ThermoFisher #11789020) was used to insert the gene into a pDONR221 plasmid (ThermoFisher #12536017). PCR mutagenesis was used to remove the N-terminal secretion sequence (Diebold et al., 2001 (link)). The resulting plasmid was linearized by PCR and HiFi DNA assembly (NEB #E2621) was used to insert an IRES sequence followed by iRFP713 (obtained from Addgene #31857) (Fig. S1 ). The pDONR plasmid was combined with the pLEX307 destination vector obtained from Addgene (#41392) and the insert transferred using LR Clonase II (ThermoFisher #11791020). The resulting pLEX307 OVA IRES iRFP713 plasmid was used to generate cell lines by viral transduction. The pMD2.G (#12259) plasmid was obtained from Addgene. The pCMV dR8.91 plasmid was generously provided by the Lim laboratory (UCSF).
Irfp713
Manufactured by Addgene
IRFP713 is a near-infrared fluorescent protein. It can be used as a reporter for visualizing biological processes in living cells and organisms.
Automatically generated - may contain errors
Lab products found in correlation
Bp clonase 2, by Thermo Fisher Scientific (2 mentions)
Lr clonase 2, by Thermo Fisher Scientific (2 mentions)
Hifi dna assembly, by New England Biolabs (2 mentions)
Pdonr221, by Thermo Fisher Scientific (2 mentions)
Plex307 destination vector, by Addgene (2 mentions)
Pmd2 g plasmid 12259, by Addgene (2 mentions)
Short tandem repeat profiling, by Promega (1 mentions)
In fusion cloning kit, by Takara Bio (1 mentions)
Mycostrip, by InvivoGen (1 mentions)
Hela cells, by RIKEN BioResource Center (1 mentions)
3 protocols using irfp713
1
Ovalbumin Protein Expression System
2
Ovalbumin Protein Expression System
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene encoding the full-length Ovalbumin protein was ordered as a synthetic gene fragment (IDT). BP Clonase II (ThermoFisher #11789020) was used to insert the gene into a pDONR221 plasmid (ThermoFisher #12536017). PCR mutagenesis was used to remove the N-terminal secretion sequence (Diebold et al., 2001 (link)). The resulting plasmid was linearized by PCR and HiFi DNA assembly (NEB #E2621) was used to insert an IRES sequence followed by iRFP713 (obtained from Addgene #31857) (Fig. S1 ). The pDONR plasmid was combined with the pLEX307 destination vector obtained from Addgene (#41392) and the insert transferred using LR Clonase II (ThermoFisher #11791020). The resulting pLEX307 OVA IRES iRFP713 plasmid was used to generate cell lines by viral transduction. The pMD2.G (#12259) plasmid was obtained from Addgene. The pCMV dR8.91 plasmid was generously provided by the Lim laboratory (UCSF).
3
Visualizing ER and Golgi dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells (RIKEN BioResource Research Center, #RCB0007) were cultured and transfected as described previously (Tojima et al., 2023 (link)). Cell line identity was verified by short tandem repeat profiling (Promega) and cells were negative for mycoplasma contamination (MycoStrip, Invivogen). Expression plasmids encoding EGFP-Sec16B, mCherry-KDEL, and EGFP-Rab1 were obtained from Addgene (#66607, #55041, and #49467 respectively). Plasmids encoding iRFP-ST, EGFP-ERGIC53, and mScarlet-I-GRASP65 under the control of CMV promoter were generated from mCherry-ST (Addgene, #55133), iRFP713 (Addgene, #31857), pMXs-IP spGFP-ERGIC53 (Addgene, #38270), EGFP-GRASP65 (Addgene, #137709), and mScarlet-I-Giantin (Addgene, #85050) using In-Fusion Cloning kit (Takara).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!