Ha tag 3724

Manufactured by Cell Signaling Technology

Sourced in United States

The HA tag (3724) is a laboratory product used for protein detection and analysis. It is a short peptide sequence derived from the hemagglutinin (HA) protein of the influenza virus. The HA tag serves as a molecular tag or label that can be fused to a target protein, allowing for its identification and purification.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Ab109250, by Abcam (1 mentions) Ab59776, by Abcam (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions) Ab134175, by Abcam (1 mentions) Neurobasal medium, by Thermo Fisher Scientific (1 mentions) Cell culture plates with ultralow attachment surfaces, by Corning (1 mentions) B27 supplement, by Thermo Fisher Scientific (1 mentions) Hct 15 cell line, by American Type Culture Collection (1 mentions) Hacat, by American Type Culture Collection (1 mentions) Ecl reagent, by Beyotime (1 mentions) C myc 10828 1 ap, by Proteintech (1 mentions) Sk mel 28, by American Type Culture Collection (1 mentions) Mg132, by Selleck Chemicals (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Propidium iodide, by BD (1 mentions)

Spelling variants of this product

HA-tag (191 citations) HA (3724) (11 citations) Rabbit anti-HA tag (3724; (3 citations)

11 protocols using ha tag 3724

Culturing and Analyzing Stem Cells

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Fetal bovine serum, trypsin, and Hank’s balanced salt solution were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture plates with ultralow attachment surfaces were purchased from Corning Life Sciences (Tewksbury, MA, USA). Neurobasal medium, FBS, B-27 Supplement, penicillin, and streptomycin were purchased from Life Technologies (Grand Island, NY, USA). TrypLE cell detachment solution was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The HCT-15 cell line was obtained from the American Type Culture Collection. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from EMD Millipore (Billerica, MA, USA). M2 anti-FLAG antibodies were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Antibodies against P53 (SC-6243) and OCT3/4 (SC-8628) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Antibodies against cyclin D1 (ab134175), NANOG (ab109250), SOX2 (ab59776), and TRRAP (ab73546) were purchased from Abcam (Boston, MA, USA). Antibodies against CD44 (5640), MYC-Tag (2287S), and HA-Tag (3724S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Kang K.T., Shin M.J., Moon H.J., Choi K.U., Suh D.S, & Kim J.H. (2023). TRRAP Enhances Cancer Stem Cell Characteristics by Regulating NANOG Protein Stability in Colon Cancer Cells. International Journal of Molecular Sciences, 24(7), 6260.

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Immortalized Cell Lines in Melanoma Research

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All the immortalized melanoma cell lines (A375, MV3 and SK-MEL-28), immortalized human melanocyte cell line Pig1, human immortalized epidermal cells HaCat and the human embryonic renal cell line 293FT were originally purchased from ATCC (American Type Culture Collection, Rockville, MD, USA). RAI14 (ab137118), RAI14 (ab241499) and FBXO32 (ab168372) were purchased from Abcam (Cambridge, MA, USA); α-Tubulin (2146), c-MYC (#9402S) and HA-Tag (3724S) were purchased from Cell Signaling Technology (Boston, MA, USA). P21 (10355-1-AP), CDK4 (66950-1-Ig), CCND1 (60186-1-Ig), GAPDH (60004-1-Ig), FBXO32 (67172-1-Ig), RAI14 (17507-1-AP) and c-MYC (10828-1-AP) were purchased from Proteintech (Wuhan, China). MG132 (#S2619) was obtained from Selleck Chemicals (Shanghai, China). Cycloheximide (CHX, #C7698) was purchased from Sigma-Aldrich (Shanghai, China). ECL reagents was obtained from Beyotime (#P0018, Shanghai, China). RIPA lysis buffer was purchased from Beyotime (Shanghai, China). Certified Fetal Bovine Serum (FBS) was purchased from VivaCell (VivaCell, Shanghai, China). Propidium iodide (PI) was purchased from BD Biosciences. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from BD Becton, Dickinson and Company.

Xu J., Shi P., Xia F., Zhao X., Chen J., Geng R., Cui H, & Yang L. (2022). RAI14 Promotes Melanoma Progression by Regulating the FBXO32/c-MYC Pathway. International Journal of Molecular Sciences, 23(19), 12036.

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Co-Immunoprecipitation Assay for Protein Interactions

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Co-immunoprecipitation (co-IP) was performed as previously described [14 (link)–16 (link)], with antibodies for p113 (ABclonal Biotechnology Co., Ltd), GST-tag (sc-33614, Santa Cruz Biotechnology), Flag-tag (14793S), ZRF1 (12844S), BRD4 (13440S), HA-tag (3724S), His-tag (12698S), MBP-tag (2396S, Cell Signaling Technology Inc., MA). Bead-bound proteins were analyzed by Coomassie blue staining, western blot, or mass spectrometry (Wuhan Institute of Biotechnology, Wuhan, China).

Yang F., Hu A., Guo Y., Wang J., Li D., Wang X., Jin S., Yuan B., Cai S., Zhou Y., Li Q., Chen G., Gao H., Zheng L, & Tong Q. (2021). p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation. Molecular Cancer, 20, 123.

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Imaging Truncated Insulin Receptor in Hippocampal Cultures

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Primary hippocampal cultures, uninfected or infected with syn-IRβ-dTomato lentivirus, were fixed using 4% paraformaldehyde (PFA) in 1X PBS for 20 min. Immunocytochemistry was performed using a primary antibody targeted to the HA-tag present on our truncated IRβ protein (HA-Tag #3724S 1:1600, Cell Signaling Technology) in conjunction with a fluorescent secondary antibody (Alexa Fluor^® 488 #A-11070 1:200, Thermo Fisher Scientific). Cultures were imaged using a spectral camera (Nuance FX, CRi, Inc.) and a FITC dichroic mirror equipped with a long-pass emission filter (>525 nm). A series of images were acquired from 490 to 650 nm and were used to define the green and all other fluorescent signals (autofluorescence and background). The green channel was extracted from the total fluorescent signal using the Nuance algorithm (spectral library subtraction).

Frazier H.N., Anderson K.L., Maimaiti S., Ghoweri A.O., Kraner S.D., Popa G.J., Hampton K.K., Mendenhall M.D., Norris C.M., Craven R.J, & Thibault O. (2018). Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents. Neurochemical research, 44(1), 269-280.

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Western Blotting Analysis of Protein Signaling

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Tissues or cells were lysed and then denatured at 100 °C for 10 min. The protein concentration was determined by bicinchoninic acid assay. Equal amounts of protein were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore Corp), and immunoblotted with the following primary antibodies: SENP2 (YT4237) was from Immunoway; phospho-AMPKα (2535S), AMPKα (5832S), phosphor-Raptor (2083), Raptor (2280), and HA tag (3724) were from Cell Signaling Technology; G6PC (A16234) and PCK1 (A2036) were from Abclonal; FLAG tag (F1804) and β-actin (A5441) were from Sigma–Aldrich; Myc tag (E022050-02) was from EΛRTH, and PCx (16588-1-AP) was from Proteintech. The specificity and reproducibility of the used antibodies were validated.

Dou X., Zhou W.Y., Ding M., Ma Y.J., Yang Q.Q., Qian S.W., Tang Y., Tang Q.Q, & Liu Y. (2021). The protease SENP2 controls hepatic gluconeogenesis by regulating the SUMOylation of the fuel sensor AMPKα. The Journal of Biological Chemistry, 298(2), 101544.

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Quantification of Protein Expression

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Primary antibodies (TRX #2429, p-ASK1 #3765, p-JNK1/2 #9251, JNK1/2 #9252, Cleaved Caspase-3 #9664, HA-Tag #3724, GAPDH #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies (Alexa Fluor^® 488 anti-rabbit #A-11034 and anti-rabbit #sc-2004) were purchased from Invitrogen (Carlsbad, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. CONPs were purchased from the NanoScale Corporation (Manhattan, KS, USA) or synthesized as previously described [14 (link),36 (link)]. For western blotting, cells were plated in 10 cm dishes and left to attach overnight. Media was then removed and replaced with media containing 0 or 10 µM CONPs. 24 h later, cells were exposed to 0 or 5 Gy RT with cell lysates collected 24–72 h after radiation. Proteins were separated on 12.5% SDS-PAGE gels, transferred to nitrocellulose membranes using the iBlot system (Invitrogen, Carlsbad, CA, USA), and blotted following standard procedures. The chemiluminescence in the blots were recorded and quantified by selecting the same exposure duration for all the membranes using Image Lab 3.0 (Bio-Rad, Hercules, CA, USA) as described previously [35 (link)].

Wason M.S., Lu H., Yu L., Lahiri S.K., Mukherjee D., Shen C., Das S., Seal S, & Zhao J. (2018). Cerium Oxide Nanoparticles Sensitize Pancreatic Cancer to Radiation Therapy through Oxidative Activation of the JNK Apoptotic Pathway. Cancers, 10(9), 303.

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Antibody-Based Signaling Pathway Analysis

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Antibodies against phospho-ERK1/2 (4370), phospho-p38 (9215), phospho-JNK1/2 (9251), phospho-IκBα (9246), TAK1 (4505), TAB2 (Leu 330, 3745), SUMO1 (4930), and HA-tag (3724) were obtained from Cell Signaling Technology. Antibodies against ERK1/2 (201245-4A4), p38 (200782), JNK1/2 (201001), IκBα (200517) and TRAF6 (380803) were obtained from Zen Bioscience, China. Anti-β-Actin (A2228), anti-Flag (F1804), and HRP-conjugated anti-Flag (A8592) antibodies were purchased from Sigma-Aldrich. Anti-Myc (MA1-21316), HRP-conjugated anti-Myc (MA1-81357), and HRP-conjugated goat anti-rabbit IgG (31460) antibodies were purchased from Invitrogen. Antibodies against TRAF6 (SC-8409), TAB2 (N48-73, SC-398188), and GST (SC-53909) were obtained from Santa Cruz Biotechnology. The antibodies against RIP1 (610458), ubiquitin (04-263), and K63-linked ubiquitin (BML-PW0600) were purchased from BD Bioscience, Merck Millipore, and Enzo Life Science, respectively. The HRP-conjugated anti-mouse secondary antibody (KCB002) was purchased from Rockland.
LPS (E. coli O127:B8, L3129), MG132 (474790), polybrene (H9268) and chloroquine (C6628) were purchased from Sigma-Aldrich. CpG-ODN 1826 (tlrl-1826) and puromycin (540411) were obtained from InvivoGen and Merck Millipore, respectively. Murine TNFα (AF-315-01A) was purchased from PeproTech.

Gu Z., Chen X., Yang W., Qi Y., Yu H., Wang X., Gong Y., Chen Q., Zhong B., Dai L., Qi S., Zhang Z., Zhang H, & Hu H. (2020). The SUMOylation of TAB2 mediated by TRIM60 inhibits MAPK/NF-κB activation and the innate immune response. Cellular and Molecular Immunology, 18(8), 1981-1994.

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Comprehensive Antibody Inventory for Research

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Antibodies against USP4 (#2651), ERK1/2 (#4695), phospho-ERK1/2 (#4370), CrkII (#3492), phospho-CrkII (#3491), PARP (#9532), cleaved-PARP (#5625), cleaved-caspase3 (#9661), HA-tag (#3724), Myc-tag (#2276), Flag-tag (#14793), rabbit mAb IgG (#3423), and mouse mAb IgG (#3420) were purchased from Cell Signaling Technology (USA). Antibodies targeting CypA (ab41684, ab58144), MMP-9 (ab38898), and Ki-67 (ab15580) were purchased from Abcam (UK). Antibodies against USP4 (for Co-IP and IHC, sc-376000), p38 (sc-7972), p-p38 (sc-7973), JNK (sc-7345), P-JNK (sc-6254) were purchased from Santa Cruz Biotechnology (USA). Anti-β-actin (AF0003), anti-GAPDH (AF0006), anti-mouse IgG (HRP) (A0216), and anti-rabbit IgG (HRP) (A0208) antibodies were purchased from Beyotime Biotechnology (China).

Li T., Yan B., Ma Y., Weng J., Yang S., Zhao N., Wang X, & Sun X. (2018). Ubiquitin-specific protease 4 promotes hepatocellular carcinoma progression via cyclophilin A stabilization and deubiquitination. Cell Death & Disease, 9(2), 148.

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Protein Immunoprecipitation and Western Blot Analysis

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Protein extracts for immunoprecipitation were prepared using nondenaturing immunoprecipitation buffer supplemented with protease and phosphatase inhibitor cocktails (78440, Thermo Fisher Scientific) and 20 mM N-ethylmaleimide (E3876, Sigma-Aldrich). Following protein quantifications, primary antibodies against His-Tag (12698, Cell Signaling Technology), Smad3 (sc-101154, Santa Cruz Biotechnology), or GSH (101-A, Virogen) were added to immunoprecipitate indicated proteins using Protein A Magnetic Beads (S1425S, New England Biolabs). The beads were washed, and protein elutes were analyzed by Western blotting. For Western blotting, cells or homogenized tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer. Primary antibodies for Western blotting include GLRX (ab187507), Smad3 (ab28379), and TGFBR1 (ab235178) from Abcam; α-SMA (A2547) from Sigma-Aldrich; Timp-1 (sc-21734) and Col1a1 (sc-293182) from Santa Cruz Biotechnology; HA-Tag (3724), His-Tag (12698), and p-Smad3 (9520) from Cell Signaling Technology; and ZFYVE9 (PA5-67946) and TGFBR2 (PA5-36115) from Invitrogen. Following incubation with secondary antibodies, Pierce ECL Substrate was used for signal detection.

Xi Y., Li Y., Xu P., Li S., Liu Z., Tung H.C., Cai X., Wang J., Huang H., Wang M., Xu M., Ren S., Li S., Zhang M., Lee Y.J., Huang L., Yang D., He J., Huang Z, & Xie W. (2021). The anti-fibrotic drug pirfenidone inhibits liver fibrosis by targeting the small oxidoreductase glutaredoxin-1. Science Advances, 7(36), eabg9241.

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Comprehensive Protein Profiling Workflow

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Whole-cell lysates were prepared using CelLytic M Cell Lysis Reagent (Sigma Aldrich, St Louis, MO, USA, C2978) or RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton-X, 0.5% sodium deoxycholate, and 0.1% SDS). The transferred membranes were incubated overnight at 4 °C with the following antibodies (catalog numbers in parentheses): HA-tag (3724), E-Cadherin (3195), N-Cadherin (13116), SNAIL (3879), Claudin-1 (13255), GFP (2956), COL1A1 (72026), and GAPDH (2118) purchased from Cell Signaling (Danvers, MA, USA); FLAG-tag (F1804), Monoclonal Anti-Flag M2 peroxidase (HRP) antibody (A8592), RIT1 (HPA053249), and β-actin (A5316) from Sigma Aldrich; LZTR1 (sc-390166) and NRAS (sc-31) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); MRAS (ab176570) and RAS (ab52939) from Abcam (Cambridge, UK); and V5-tag (M215-3), HRAS (18295-1-AP), and KRAS (OP24) from MBL International (MA, USA), Proteintech (Chicago, IL, USA), and Merck Millipore (Billerica, MA, USA), respectively. Original western blots showed in this study are provided in Supplementary Fig. S5.

Abe T., Kanno S.I., Niihori T., Terao M., Takada S, & Aoki Y. (2023). LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and controlling KLHL12-mediated collagen secretion. Cell Death & Disease, 14(8), 556.

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