Sfgfp c1

Manufactured by Addgene

SfGFP-C1 is a plasmid that contains the gene encoding superfolder green fluorescent protein (sfGFP). It is commonly used as a fluorescent protein reporter for various research applications.

Automatically generated - may contain errors

Lab products found in correlation

Taok2 cdna, by OriGene (2 mentions) Nebuilder hifi dna assembly, by New England Biolabs (1 mentions) Ecori, by New England Biolabs (1 mentions) Crisprv2pspcas9 bb 2a puro px459 v2, by Addgene (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Calreticulin, by Abcam (1 mentions) Acetylated α tubulin, by Merck Group (1 mentions) Tom20, by Santa Cruz Biotechnology (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Stim1, by Santa Cruz Biotechnology (1 mentions) Alpha tubulin, by Merck Group (1 mentions) Er mrfp, by Addgene (1 mentions)

Close spelling variants of this product

SfGFP

6 protocols using sfgfp c1

Cloning and Characterization of TAOK2 Domains

Check if the same lab product or an alternative is used in the 5 most similar protocols

Full-length human TAOK2 was PCR amplified from TAOK2 cDNA (Origene, isoform alpha), and inserted in vector sfGFP-C1 (Addgene #54579) using restriction sites HindIII and MfeI. Domain dissection mutants were subcloned from sfGFP-TAOK2 using restriction enzymes HindIII and MfeI (New England Biolabs). All resultant plasmids were verified by sequencing. GST-TAOK2-(1187–1235) was subcloned from the sfGFP-TAOK2 into the pGEX4T1 vector using sites SalI and NotI. BFP-STIM1 was created by subcloning BFP from BFP-Rab5 (#49147) and inserting into STIM-YFP (#19754) using restriction sites SalI and NotI.

Nourbakhsh K., Ferreccio A.A., Bernard M.J, & Yadav S. (2021). TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubules. Developmental cell, 56(24), 3321-3333.e5.

+ Open protocol

+ Expand

Cloning and Characterization of TAOK2 Domains

Check if the same lab product or an alternative is used in the 5 most similar protocols

Nourbakhsh K., Ferreccio A.A., Bernard M.J, & Yadav S. (2021). TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubules. Developmental cell, 56(24), 3321-3333.e5.

+ Open protocol

+ Expand

Proinsulin Tracking System Generation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

ProCpepRUSH (SA-KDEL-IRES-proCpep-SBP-GFP) was generated by Gibson assembly and subcloned into pENTR2b-RIP^{12 (link)}. SA-KDEL-IRES and SBP PCR fragments were separately amplified from Str-KDEL_SBP-EGFP-GPI (gift from Franck Perez, Addgene plasmid #65,294; RRID:Addgene_65294)^{28 (link)}. sfGFP was PCR amplified from sfGFP-C1 (gift from Michael Davidson and Geoffrey Waldo, Addgene plasmid #54,579; RRID:Addgene_54579)^{65 (link)}. Proinsulin was PCR amplified as two fragments on either side of the ApaI site from human INS cDNA for insertion of the SBP and sfGFP fragments within the C-peptide coding sequence. RIP-SA-KDEL-IRES-proCpep-SBP-GFP was recombined from pENTR2b-RIP into a modified pAD-PL/DEST via Gateway cloning using LR Clonase II^{66 (link)}. Recombinant adenoviruses were generated in HEK293 cells and purified by cesium chloride gradient. All sequences were verified by the Iowa Institute of Human Genetics, University of Iowa.

Boyer C.K., Bauchle C.J., Zhang J., Wang Y, & Stephens S.B. (2023). Synchronized proinsulin trafficking reveals delayed Golgi export accompanies β-cell secretory dysfunction in rodent models of hyperglycemia. Scientific Reports, 13, 5218.

+ Open protocol

+ Expand

Cloning and Mutation of TAOK1 Construct

Check if the same lab product or an alternative is used in the 5 most similar protocols

Full length human TAOK1 was obtained from Transomics Technologies in PCR-XL-Topo plasmid and inserted into sfGFP-C1 (Plasmid #54579, Addgene), using the EcoRI and MfeI sites. The resulting N-terminally sfGFP-tagged TAOK1 construct (sfGFP-TAOK1) was fully sequenced (Genewiz, Azenta Life Sciences) and used as template to generate the ASD associated mutants (Genewiz). All constructs were confirmed by sequencing for accuracy. Domain deletion mutants were subcloned from sfGFP-TAOK1 using restriction enzymes EcoRI and MfeI (New England Biolabs) and assembled using NEBuilder HiFi DNA Assembly-New England BioLabs. Resultant plasmids were verified by sequencing. GST-TAOK1(321–901) was generated by subcloning from the sfGFP-TAOK1 into the pGEX4T1 vector using restriction enzymes NotI and SalI.

Beeman N., Sapre T., Ong S.E, & Yadav S. (2023). Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase. Science signaling, 16(766), eadd3269.

+ Open protocol

+ Expand

Generating TAOK2 Constructs and Knockouts

Check if the same lab product or an alternative is used in the 5 most similar protocols

Full length human TAOK2 was PCR amplified from pCMV-Sp6-TAOK2 plasmid described previously (Ultanir et al., 2014) , and inserted in vector sfGFP-C1 (Addgene #54579) using restriction sites HindIII and MfeI. Domain dissection mutants were subcloned from sfGFP-TAOK2 using restriction enzymes HindIII and MfeI (New England Biolabs). All resultant plasmids were verified by sequencing. GST-TAOK2-(1187 -1235) was subcloned from the sfGFP-TAOK2 into the pGEX4T1 vector using sites SalI and NotI. TAOK2 knockout cell line was generated using CRISPR/Cas9 genome editing in HEK293T cells. Four independent guides were designed using Synthego guide design tool (https://www.synthego.com) to target coding exon 2. Two plasmids were made by adding their respective guides into CrisprV2pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene Plasmid #62988). Cells were passaged in single cell suspension and plated at 50% confluence. Cultures were then transfected with lipofectamine 2000 reagent (Invitrogen 11668-030) and 2mg of each respective plasmid. Non-Homology End Joining (NHEJ) repair created a deletion around the gRNA cutting site. Cells were selected with Puromycin for 2 days. Genomic DNA was extracted and the region around the cutting site was PCR amplified. Knockout of the gene TAOK2 was confirmed by Sanger sequencing analysis, and absence of encoded protein was validated using western blot.

Nourbakhsh K., Ferreccio A., Bernard M.J., & Yadav S. (2021). TAOK2 is an ER-localized Kinase that Catalyzes the Dynamic Tethering of ER to Microtubules.

+ Open protocol

+ Expand

Antibody Characterization and Plasmid Identification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibodies used in this study are as follows: alpha-Tubulin (Mouse, Sigma, T9026-100UL), GAPDH (Mouse, Invitrogen, MA5-151738), Calreticulin (Mouse, Abcam, ab22683), TAOK2, Rabbit, Sigma, HPA010650), Rtn3a (Rabbit, ProteinTech,12055-2-AP), Acetylated alpha Tubulin (Mouse, Sigma, T6793), GST (Mouse, Invitrogen), GM130 (Mouse, BD Labs, 610822), TAOK2-Cterm (Rabbit), Tom20 (Mouse, Santa Cruz Biotech, sc-17764), Stim1 (Mouse, Santa Cruz Biotech, sc-166840), GFP (Mouse, Roche, 11 814 460 001), Phospho-TAO2 (S181) (Rabbit, R&D Systems, PPS037). Addgene Plasmids used in this study are as follows: mCh-Sec61 beta (#49155), sfGFP-C1 (#54579), ER-mRFP (#62236), EGFP-Sec22b (#101918).

Nourbakhsh K., Ferreccio A., Bernard M.J., & Yadav S. (2021). TAOK2 is an ER-localized Kinase that Catalyzes the Dynamic Tethering of ER to Microtubules.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!