Hrp conjugated anti flag antibody

Manufactured by Merck Group

Sourced in United States

The HRP-conjugated anti-FLAG antibody is a laboratory reagent used for the detection and identification of proteins tagged with the FLAG epitope. The antibody is conjugated with horseradish peroxidase (HRP), which allows for colorimetric or chemiluminescent detection of the target protein in various applications such as Western blotting, immunohistochemistry, and ELISA.

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Close spelling variants of this product

Monoclonal anti-FLAG antibody conjugated to HRP (2 citations) Mouse HRP-conjugated anti-FLAG M2 antibody (2 citations) HRP-conjugated anti-FLAG M2 antibody (19 citations) Horseradish peroxidase (HRP)-conjugated anti-FLAG antibody (2 citations) Anti-FLAG M2 HRP-conjugated monoclonal antibody (3 citations) HRP-conjugated mouse anti-FLAG tag antibody (3 citations) HRP-conjugated anti-FLAG tag antibody (4 citations) HRP-conjugated anti-FLAG (17 citations) Anti-FLAG-HRP antibody (18 citations) Anti-FLAG-HRP (103 citations)

37 protocols using hrp conjugated anti flag antibody

Characterization of Leukocyte Adhesion

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Mouse anti-FLAG M2 antibody (Sigma), HRP-conjugated anti-FLAG antibody (Sigma), APC PSGL-1 antibody (FLEG) (ThermoFisher), Human Fibronectin (ThermoFisher), DMSO (Sigma), Phalloidin Alex647 (ThermoFisher), RIPA buffer (Sigma), Phorbol 12-Myristate 13-Acetate (PMA, sigma), SLeX (Sigma), Biotinylated ECL (Vector Laboratories), PNGase F (New England Biolabs), Dihydrorhodamine 123 (DHR123) (ThermoFisher), ProLong Gold Antifade Mountant (ThermoFisher), HRP-conjugated streptavidin (ThermoFisher).

Chen C., Yang C, & Barbieri J.T. (2019). Staphylococcal Superantigen-like protein 11 mediates neutrophil adhesion and motility arrest, a unique bacterial toxin action. Scientific Reports, 9, 4211.

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Co-immunoprecipitation of GmGAMYB and GmGBP1

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The constructed GmGBP1-TOPO was transferred to the expression vector pGWB506 by LR reaction (named 35S:GmGBP1-GFP). The constructed recombinant was introduced into Agrobacterium GV3101. Agrobacterium 35S:GmGAMYB-3F6H-pB7WG2 and 35S:GmGBP1-GFP were individually or collectively transformed into N. Benthamiana leaves (Hu et al., 2013 (link)) and were sampled 2 days later. After protein extraction with a Co-IP buffer [50 mM Na-phosphate pH7.4, 135 mM NaCl, 4.7 mM KCl, 1 mM DTT, 50 μM MG-132, 2 mM Na₃VO₄, 2 mM NaF, and Complete protease inhibitor cocktail tablets (Roche)], 10 μl of Protein G-coupled magnetic beads (Dynabeads Protein G, Invitrogen) was used to capture anti-FLAG antibody (Sigma). After incubation at 4°C for 30 min, magnetic beads were washed three times for 5 min each time with 1 ml of Co-IP buffer without MG-132, Na₃VO₄, sodium fluoride, and protease inhibitor, and eluded with 2 × SDS sample buffer at 80°C for 5 min. At each sample, the immunoprecipitated proteins and 20 μl of the total extract were separated by 10% SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. Then, HRP-conjugated anti-FLAG antibody (A8592, Sigma) and HRP-conjugated anti-GFP Antibody (AB6663, Abcam) were applied to test GmGAMYB-FLAG and GmGBP1-GFP protein, respectively.

Yang X., Li X., Shan J., Li Y., Zhang Y., Wang Y., Li W, & Zhao L. (2021). Overexpression of GmGAMYB Accelerates the Transition to Flowering and Increases Plant Height in Soybean. Frontiers in Plant Science, 12, 667242.

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Arabidopsis Protein Extraction and Co-IP

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Total Arabidopsis protein was extracted from 0.5 g of ground powder in 2 mL of extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % Triton X-100, 5 mM EDTA, 1x Protease inhibitor cocktail [Sigma-Aldrich] and 1 mM PMSF) and centrifuged at 4 °C at maximum speed for 10 min. 50 μL input was taken from the supernatant and 1 mL protein extract were incubated with 20 μL of anti-FLAG-M2-Agarose beads (Sigma-Aldrich A2220) for 1.5 h at 4 °C. And then washed three times with wash buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20). Samples were analyzed by SDS-PAGE and immunoblotting using HRP-conjugated anti-FLAG antibody (Sigma-Aldrich) and anti-H2A antibody (Abcam) as described above.
To detect the PIF4-RGA-H2A or IDD3-RGA-H2A complex, MYC-PIF4 or MYC-IDD3, HA-RGA and FLAG-H2A were transiently expressed in N. benthamiana leaves, and subsequent co-IP assays using rabbit anti-Myc polyclonal antibody conjugated agarose beads (Sigma-Aldrich A7470) were performed as described previously^{45 (link)}.

Hébert P.C., Levin A.V, & Robertson G. (2001). The error of our ways. CMAJ: Canadian Medical Association Journal, 165(3), 271-272.

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Binding Assay of Fc Mutants

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ELISA was used to determine the binding capability of the selected proteins to various unrelated antigens. All Fc mutants (mFc, 1-B10-9, 1-D1-15) were expressed in E. coli HB2151, as described above. Antigens (gp140, mesothelin, ZIKV EDII, ZIKV EDIII, 5T4, PD-L1, OX40, TIM-3) were coated on 96-well ELISA plates (Corning, #3690) overnight with 100 ng/well in PBS at 4°C, and blocked with 100 µl per well of 3% MPBS (PBS with 3% milk) at 37°C for 1 h. The plates were washed with PBS with 0.05% Tween 20 (PBST), then threefold serial diluted proteins were added and incubated at 37°C for 1.5 h. Plates were washed five times with PBST and 50 µl of 1:1,000 HRP conjugated anti-FLAG antibody (Sigma-Aldrich) in PBS were added per well before incubation at 37°C for 45 min. After extensive washes with PBST, the binding was measured by the addition of diammonium 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) substrate (Roche Applied Science) and signal reading was carried out at 405 nm.

Wang C., Wu Y., Wang L., Hong B., Jin Y., Hu D., Chen G., Kong Y., Huang A., Hua G, & Ying T. (2017). Engineered Soluble Monomeric IgG1 Fc with Significantly Decreased Non-Specific Binding. Frontiers in Immunology, 8, 1545.

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Nanobody Binding to LASV Glycoprotein

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Protein ELISA was used to evaluate the binding ability of the selected nanobody binders toward the stabilized LASV GPC trimer. Briefly, a 96-well plate was coated with either the stabilized LASV GPC trimer or BSA at 5 μg/ml in PBS, 50 μl/well, at 4 °C overnight. After blocking with 100% superblock buffer, the nanobodies were diluted into 1 μg/ml using 10% PBST in 100% superblock and then added to the plate for incubation at room temperature for 1 h. Binding signal was detected by HRP-conjugated anti-Flag antibody (Sigma). For the competition ELISA, neutralizing antibodies D5, 19.7E, 8.11 G, and 37.7H were used to block the binding epitopes. An anti-Foldon antibody, MF4 were also used as a control.

Gorman J., Cheung C.S., Duan Z., Ou L., Wang M., Chen X., Cheng C., Biju A., Sun Y., Wang P., Yang Y., Zhang B., Boyington J.C., Bylund T., Charaf S., Chen S.J., Du H., Henry A.R., Liu T., Sarfo E.K., Schramm C.A., Shen C.H., Stephens T., Teng I.T., Todd J.P., Tsybovsky Y., Verardi R., Wang D., Wang S., Wang Z., Zheng C.Y., Zhou T., Douek D.C., Mascola J.R., Ho D.D., Ho M, & Kwong P.D. (2024). Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability. Nature Communications, 15, 285.

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Yeast Rapamycin Response Assay

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Yeast grown in 5 mL synthetic complete media with or without rapamycin treatment were collected when OD600 reached 0.5. Yeast pellet was lysed with 200 μL of glass beads in 200 μL buffer containing 25 mM Tris at pH=8.0 and 150 mM NaCl. 1 μg of total yeast lysate was used for western blot against HRP-conjugated anti-Flag antibody (Sigma A8592).

Zhang Y., Lin Z., Wang M, & Lin H. (2018). Selective usage of isozymes for stress response. ACS chemical biology, 13(11), 3059-3064.

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Production and Purification of scFv Protein in P. pastoris

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For production of scFv protein, P. pastoris X33 cells, harboring the multicopy integrants, were grown overnight at 30°C in 50 mL BMGY (Buffered Glycerol-complex medium; 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, and 1.34% YNB (Yeast Nitrogen Base, Invitrogen), 4×10⁻⁵% biotin and 1% glycerol) at 300 rpm. The next day, the cells were pelleted (2500 rpm, 10 min, RT), resuspended in 200 mL BMMY (Buffered Methanol-complex medium: 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4×10⁻⁵% biotin and 0.5% methanol), and grown for three days at 300 rpm; supplementing methanol (25 mL; 0.5%) was added each day to compensate for loss by evaporation. The culture medium was collected after the third day by centrifugation of cultures (2500 rpm, 10 min, RT), and the secreted scFvs were isolated by immobilized metal affinity chromatography, using Ni-NTA conjugated agarose beads (Qiagen) and elution with PBS containing 200 mM imidazole (Sigma). The purified proteins were diluted 1∶50 in PBS, tested by ELISA as described above, and detected using HRP conjugated anti-Flag antibody (diluted 1∶10,000 in PBS; Sigma).

Koduvayur S.P., Gussin H.A., Parthasarathy R., Hao Z., Kay B.K, & Pepperberg D.R. (2014). Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides. PLoS ONE, 9(2), e87964.

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Quantifying Surface Expression of Mutant Receptors

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Surface expression levels of WT and mutated hGCGR variants were tracked using a direct enzyme-linked immunosorbent assay (ELISA) against the N-terminal FLAG tag. In brief, cells were transfected and seeded in a white Poly-D-Lysine-coated 96-well plate as described for the functional cAMP accumulation assay. Twenty-four hours after transfection, cells were washed with 100 μl/well DPBS + 1 mM calcium chloride and fixed with 50 μl/well 4% paraformaldehyde solution for 5 min at room temperature. The wells were washed twice with 100 μl DPBS + 1 mM calcium chloride and blocked with 100 μl/well blocking solution (3% drymilk, 1 mM calcium chloride 50 mM Tris-HCl, pH 7.5) for 30 min at room temperature. Hereafter 75 μl/well HRP-conjugated anti-FLAG antibody (Sigma Aldrich, A8592), diluted 1:2000 in blocking solution, was added and the plate was incubated for 1 hour at room temperature. The plate was washed four times with 100 μl/well blocking solution followed by four washes with 100 μl/well DPBS + 1 mM calcium chloride. The relative amount of present HRP was detected by adding 60 μl/well DPBS + 1 mM calcium chloride and 20 μl/well HRP substrate (Bio-Rad, 170–5060). The plate was incubated for 10 min at room temperature before recording of luminescence on an EnVision plate reader (Perkin Elmer).

Hilger D., Kumar K.K., Hu H., Pedersen M.F., O’Brien E.S., Giehm L., Jennings C., Eskici G., Inoue A., Lerch M., Mathiesen J.M., Skiniotis G, & Kobilka B.K. (2020). Structural insights into differences in G protein activation by family A and family B GPCRs. Science (New York, N.Y.), 369(6503), eaba3373.

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Western Blot Analysis of FLAG-Tagged Proteins

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Two μg of total protein per sample was loaded onto the NuPAGE 4%–12% Bis-Tris gel (Invitrogen# NP0321), along with 10 μL of Spectra Multicolor Protein Ladder (Thermo# 26634). Proteins were resolved at 200 V for 50 min in MOPS SDS running buffer. Following resolution, proteins were transferred to PVDF membrane (Invitrogen# LC2005) at 100 V for 60 min. The membrane was blocked in non-fat milk for 1 h to reduce non-specific binding. The blot was then probed with HRP conjugated anti-FLAG antibody (Sigma# A8592) at 1:2000 through overnight incubation at 4°C. The membrane was then washed multiple times with TBS buffer, supplemented with 0.1% Tween 20, to wash off excess unbound antibodies. SuperSignal West Femto Substrate (Thermo# 34095) was used to develop the blot, and it was imaged in the iBright FL1500 Imaging system (Thermo Fisher). A second NuPAGE gel, run with the same samples, was stained with SYPRO Ruby Protein Gel Stain (Invitrogen# S12001) following the manufacturer’s protocol and imaged to confirm equal loading.

Webster A.K., Chitrakar R., Taylor S.M, & Baugh L.R. (2022). Alternative somatic and germline gene-regulatory strategies during starvation-induced developmental arrest. Cell reports, 41(2), 111473.

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Immunoprecipitation of Sirt1 and Interactors

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10.10⁶ cells were harvested, washed with PBS, and resuspended in lysis buffer (400 mM NaCl, 10 mM HEPES, pH 7.5, 0.1% NP‐40, Sigma P8340); the NaCl concentration was then brought down to 150 mM; and samples were sonicated using a probe sonicator and then centrifuged at 17,000 rcf in order to remove insoluble material. The protein concentration of the lysates was measured using a BCA assay (Thermo Fisher, 23225), and equivalent protein quantities were then incubated with 50 μl of pre‐washed anti‐HA agarose beads (Sigma, A2095) overnight on a rotating wheel at 4°C. The samples were then incubated five times for 10 min with 1 ml of wash buffer (150 mM NaCl, 10 mM HEPES, pH 7.9, 0.1% NP‐40, protease inhibitors) on a rotating wheel at 4°C. Immunoprecipitates were eluted in Laemmli buffer at 95°C for 10 min. Similar elution volumes and protein quantities, for the IPs and the inputs, respectively, were loaded on a gel. Western blots were performed using anti‐Sirt1 (Abcam, ab32441), anti‐IPO7 (Abcam, ab99273), anti‐GFP (Abcam, ab5450), anti‐KAP1 (Merck, MAB3662), HRP‐conjugated anti‐Flag antibody (Sigma, A8592), HRP‐conjugated anti‐HA antibody (Sigma, 12013819001), HRP‐conjugated anti‐goat antibody (Dakocytomation, P0449), HRP‐conjugated anti‐rabbit antibody (Santa Cruz, sc‐2004), and HRP‐conjugated anti‐mouse antibody (GE Healthcare, NA931V).

Helleboid P., Heusel M., Duc J., Piot C., Thorball C.W., Coluccio A., Pontis J., Imbeault M., Turelli P., Aebersold R, & Trono D. (2019). The interactome of KRAB zinc finger proteins reveals the evolutionary history of their functional diversification. The EMBO Journal, 38(18), e101220.

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