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Flag tag (flag)

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FLAG is a molecular tag used for the identification and purification of recombinant proteins. It consists of a short peptide sequence that can be fused to the target protein, allowing for detection and isolation using specific antibodies or affinity chromatography.

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Lab products found in correlation

C myc, by Novus Biologicals (4 mentions) Mcherry, by Takara Bio (3 mentions) Gfp trap reagent, by Proteintech (3 mentions) Mphosph8, by Proteintech (2 mentions) Pphln1, by Merck Group (2 mentions) Morc2, by Fortis Life Sciences (2 mentions) Setdb1, by Proteintech (2 mentions) Dcaf1, by Proteintech (2 mentions) Line 1 orf1p, by Merck Group (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Appropriate secondary anti mouse or anti rabbit hrp conjugate, by Cell Signaling Technology (2 mentions) Antifade mounting medium, by Vector Laboratories (1 mentions) Fluorescein isothiocyanate (fitc), by Santa Cruz Biotechnology (1 mentions) Tritc, by Santa Cruz Biotechnology (1 mentions) Rfp trap, by Proteintech (1 mentions) Dmem media, by Merck Group (1 mentions) Zen black 2012, by Zeiss (1 mentions) Alexa fluor 594, by Thermo Fisher Scientific (1 mentions) L glutamin, by Thermo Fisher Scientific (1 mentions) Fugene 6 reagent, by Promega (1 mentions)

8 protocols using flag tag (flag)

1

Western Blot Analysis of Protein Markers

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Cells were washed in PBS, counted, normalized for cell number, and lysed directly in 1x SDS-PAGE sample buffer. Samples were run on NuPage 4–12% Bis-Tris gels followed by blotting onto nitrocellulose membranes. Primary antibodies used: FAM208A (Atlas, HPA00875), MPHOSPH8 (Proteintech, 16796–1-AP), PPHLN1 (Sigma, HPA038902), SETDB1 (Proteintech 11231–1-AP), DCAF1 (Proteintech, 11612–1-AP), FLAG (Novus, NB600–345), FLAG (Sigma, F1804, used for IP), MORC2 (Bethyl Labs, A300–149A), LINE-1 ORF1p (Millipore, MABC1152),and HA (Biolegend, 901501).
Yurkovetskiy L., Guney M.H., Kim K., Goh S.L., McCauley S., Dauphin A., Diehl W, & Luban J. (2018). Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex. Nature microbiology, 3(12), 1354-1361.
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2

Immunofluorescence Staining of Epitope-Tagged Proteins

Cited 1 time
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Cell monolayers were washed with PBS and fixed in methanol overnight at −20°C. Cell were rehydrated in PBS (3× 5min) prior to blocking in 5% Goat serum/PBST at room temperature. Cell were incubated for 1h at room temperature with specific primary antibodies targeting epitope tags; HA (Novus Biologicals); FLAG (Novus Biological); and myc-c (Novus Biologicals) antibodies at a concentration of 100 ng/ml as was antibody targeting Pan-keratin (C11) (Cell Signaling). Use of rabbit anti-GPCMV IE2 and mouse anti-GP133 was as previously described in (67 (link)). Cells were washed and incubated for 1h with secondary antibodies, either goat anti-mouse or goat anti-rabbit, congregated with FITC or TRITC (Santa cruz). Cells were counter stained with DAPI prior to mounting in anti-fade mounting medium (Vectashield).
Coleman S., Choi K.Y, & McGregor A. (2017). Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig. Virology, 509, 205-221.
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3

Epitope-Tagged Protein Immunoprecipitation

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Immunoprecipitation (IP) assays were carried out on plasmid transfected or recombinant Ad transduced fibroblast cells using commercial GFP-trap reagent (ChromoTek) or RFP-trap (ChromoTek) following manufacturer’s protocols and inclusion of protease inhibitor cocktail (Pierce) in cell lysates. Samples were subsequently analyzed by SDS-PAGE (4–20% gradient gel) and western blot using specific anti-epitope tag antibodies: HA (Novus Biologicals); FLAG (Novus Biological); GFP (Santa Cruz Biotechnology); Myc-c (Novus Biologicals); and mCherry (Clontech Laboratories). Appropriate secondary anti-mouse or anti-rabbit HRP conjugate (Cell Signaling Technology) were also used following standard western blot protocol as previously described [36 (link)].
Coleman S., Choi K.Y., Root M, & McGregor A. (2016). A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus. PLoS Pathogens, 12(7), e1005755.
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4

Immunofluorescence Localization of MORN5 Protein

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The expression vector containing C-terminally FLAG-tagged human MORN5 was obtained from OriGene (Rockville, MD). HEK293T cells were obtained from ATCC (Manassas, VA) and propagated in DMEM media (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum, 1% Pen/Strep and 1% l-glutamin (Invitrogen, Carlsbad, CA). Cells were transfected using FUGENE6 reagent according to manufacturer's protocol (Promega).
HEK293T cells grown on glass coverslips were fixed with 4% PFA (RT/15 min), permeabilized with 0.1%Triton-X100 in PBS (RT/5 min), and incubated with the following antibodies at 4°C overnight: MORN5 (1:100, cat. No. NBP1-91230, Novus Biologicals), FLAG (1:200, cat. No. F1804, Sigma-Aldrich). The secondary antibody AlexaFluor 488 (1:500; cat. No. A21206, Life Technologies) or AlexaFluor 594 (1:500; cat. No. A21203, Life Technologies) were used. Coverslips were mounted into DAPI-containing Mowiol. Images were taken on an LSM700 laser scanning microscope with acquisition done using ZEN Black 2012 software (Zeiss, Jenna, Germany).
Cela P., Hampl M., Fu K.K., Kunova Bosakova M., Krejci P., Richman J.M, & Buchtova M. (2016). MORN5 Expression during Craniofacial Development and Its Interaction with the BMP and TGFβ Pathways. Frontiers in Physiology, 7, 378.
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5

Western Blot Analysis of Protein Markers

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Cells were washed in PBS, counted, normalized for cell number, and lysed directly in 1x SDS-PAGE sample buffer. Samples were run on NuPage 4–12% Bis-Tris gels followed by blotting onto nitrocellulose membranes. Primary antibodies used: FAM208A (Atlas, HPA00875), MPHOSPH8 (Proteintech, 16796–1-AP), PPHLN1 (Sigma, HPA038902), SETDB1 (Proteintech 11231–1-AP), DCAF1 (Proteintech, 11612–1-AP), FLAG (Novus, NB600–345), FLAG (Sigma, F1804, used for IP), MORC2 (Bethyl Labs, A300–149A), LINE-1 ORF1p (Millipore, MABC1152),and HA (Biolegend, 901501).
Yurkovetskiy L., Guney M.H., Kim K., Goh S.L., McCauley S., Dauphin A., Diehl W, & Luban J. (2018). Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex. Nature microbiology, 3(12), 1354-1361.
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6

Analyzing Protein-Protein Interactions

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Immunoprecipitation (IP) assays were carried out on plasmid transfected or recombinant Ad transduced fibroblast cells using commercial GFP-trap reagent (ChromoTek) following manufacturer’s protocol and inclusion of protease inhibitor cocktail (Pierce) in cell lysates. Samples were subsequently analyzed by SDS-PAGE (4–20% gradient gel) and western blot using specific anti-epitope tag antibodies: HA (Novus Biologicals); FLAG (Novus Biological); GFP (Santa Cruz); Myc-c (Novus Biologicals); and mCherry (Clontech). Appropriate secondary anti-mouse or anti-rabbit HRP conjugate (Cell Signaling Technology) were also used following standard western blot protocol.
Coleman S., Hornig J., Maddux S., Choi K.Y, & McGregor A. (2015). Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus. PLoS ONE, 10(8), e0135567.
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7

Immunoprecipitation Assays of Pentameric Components

Cited 2 times
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Immunoprecipitation (IP) assays were carried out on plasmid transfected renal epithelial cells using commercial GFP-trap reagent (ChromoTek) following manufacturer’s protocols and as previously described (67 (link)). Samples were subsequently analyzed by SDS-PAGE (4–20% gradient gel) and western blot using specific anti-epitope tag antibodies: HA (Novus Biologicals); FLAG (Novus Biological); GFP (Santa Cruz Biotechnology); myc-c (Novus Biologicals); and mCherry (Clontech Laboratories). Appropriate secondary anti-mouse or anti-rabbit HRP conjugate (Cell Signaling Technology) were also used following standard western blot protocol as previously described (68 (link)). GP129 C-terminus deletion mutants IP assays were performed using epitope tagged pentameric components (gHGFP, gLmCherry, GP131HA and GP133FLAG) transfected with either GP129DEL1, GP129DEL2 or GP129DEL3. IP studies of GP131d120 and GP133d75 used the same pentameric component plasmids substituting GP131HA with GP131d120 or GP133FLAG with GP133d75. Full length GP129 was used in both IP assays as positive control (67 (link)). Control IP used GFP plasmid transfected with gLmCherry, GP129myc, GP131HA and GP133FLAG to verify gH specific binding (Fig 5).
Coleman S., Choi K.Y, & McGregor A. (2017). Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig. Virology, 509, 205-221.
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8

Antibody and Reagent Inventory for Cellular Analysis

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Antibodies were obtained as follows: α‐tubulin (Cat No. AF5012; Beyotime), TIM23 (Cat No. sc‐514463; Santa Cruz Biotechnology), GFP‐Trap® Agarose (Cat No. gta; ChromoTek), anti‐FLAG® M2 affinity gel (Cat No. A2220; Sigma–Aldrich), m6A (Cat. No. 56593; CST), TOM20 (Cat No. 42406; CST) and COXIV (Cat No. 11967; CST).
All of these antibodies were purchased from Proteintech: glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Cat No. 60004‐1‐Ig), FLAG (Cat No. 20543‐1‐AP), FSCN1 (Cat No. 14384‐1‐AP), GFP (Cat No. 50430‐2‐AP), IGF2BP1 (Cat No. 22803‐1‐AP), IGF2BP2 (Cat No. 11601‐1‐AP), IGF2BP3 (Cat No. 14642‐1‐AP), Ki67 (Cat No. 27309‐1‐AP), MYC (Cat No. 10828‐1‐AP), MARCKSL1 (Cat No. 10004‐2‐Ig), LC3 (Cat No. 14600‐1‐AP), Parkin (Cat No. 14060‐1‐AP), TK1 (Cat No. 15691‐1‐AP), ubiquitin (Cat No. 10201‐2‐AP), VDAC1 (Cat No. 10866‐1‐AP).
Recombinant proteins were purchased from Novus: ubiquitin‐activating Enzyme/UBE1 (Cat No. E‐305), UbcH7/UBE2L3 (Cat No. E2‐640), ubiquitin (Cat No. U‐100H), Parkin (Novus, E3‐160). Recombinant IGF2BP3 protein was purchased from Abnova (Cat No. H00010643‐P01).
All chemicals were purchased from MedChemExpress: actinomycin D (Cat No. HY‐17559), CCCP (Cat No. HY‐100941), MG132 (Cat No. HY‐13259), CHX (Cat No. HY‐12320), SAHA (Cat No. HY‐10221).
Sun X., Ye G., Li J., Shou H., Bai G, & Zhang J. (2023). Parkin regulates IGF2BP3 through ubiquitination in the tumourigenesis of cervical cancer. Clinical and Translational Medicine, 13(10), e1457.
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