Protein extracts were heated for 5 min at 95 °C, loaded into 4–12% Bis-Tris gels (Life Technologies), and run at 150V. Proteins were transferred into immobilon-P PVDF membranes at 400 mA, blocked in 5% milk in TBS-Tween 0.1% (TBST), and incubated with primary antibodies overnight at 4 °C. All secondary HRP-conjugated antibodies were incubated for 1 h at room temperature. The following primary antibodies were used: Anti-HA-Tag (Cell Signaling, C29F4), Anti-FLAG® (Sigma-Aldrich, F1804), Anti-DYKDDDDK Tag (Cell Signaling, D6W5B), anti-β-actin (Santa Cruz, sc-47778), anti-α-tubulin (Licor, 926-42213), anti-vinculin (Sigma-Aldrich, V9131), anti-hTREM2 (R&D AF1828), anti-TREM2222 (Ab222), and anti-TREM2219 (Ab219). Ab222 and Ab219 are custom monospecific antibodies generated by Pacific Immunology. The antigen for Ab222 is CSLAWTEARDTSTQ, and for Ab219 is RAERHVKEDDGRKSPGEVPPGTS-Cys. Rabbits were immunized, and their serum was used to purify antibodies by affinity purification against the above mentioned protein sequences. This process allows the isolation of highly specific antibodies that approach the specificity of monoclonal antibodies and provide the superior affinity of polyclonal antibodies (https://www.pacificimmunology.com ). All antibodies were diluted in 5% milk in TBST, except for anti-vinculin which was dissolved in 5% bovine serum albumin (BSA) in TBST.
Anti dykddddk tag
Manufactured by Cell Signaling Technology
Sourced in China, United States
The Anti-DYKDDDDK Tag is a laboratory reagent used to detect and purify proteins tagged with the DYKDDDDK sequence, also known as the 'FLAG' tag. It can be used in various applications such as immunoprecipitation, Western blotting, and affinity chromatography to identify and isolate tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti myc tag, by Merck Group (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Immobilon p e pvdf membrane, by Merck Group (2 mentions)
Anti vinculin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti ha tag, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate kit, by Merck Group (1 mentions)
A0216, by Beyotime (1 mentions)
Goat anti mouse igg, by Beyotime (1 mentions)
Ap0063, by Bioworld Technology (1 mentions)
Ab223175, by Abcam (1 mentions)
Ab167180, by Abcam (1 mentions)
Ab9110, by Abcam (1 mentions)
6 protocols using anti dykddddk tag
1
Western Blot Antibody Characterization
2
Protein Expression and Immunoblotting
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Cultured cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China). BCA kit (Pierce, Rockford, IL) was used to measure protein concentrations. The Flag and HA sequences were inserted into plasmids by the primers containing specific restriction sites. The plasmids were transformed into competent cells, screened, and amplified with LB containing ampicillin. High-concentration recombinant Flag-TMED3 and HA-FAM60A were obtained. These two plasmids were verified by DNA sequencing. Endotoxin-free extraction was carried out for subsequent cell transfection [18 (link)]. The equivalent amounts of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA). PVDF membrane was blocked with a blocking buffer and was incubated with an anti-TMED3 (ab223175, Abcam), anti-FAM60A (ab167180, Abcam), anti-GAPDH (AP0063, Bioworld, Nanjing, China), anti-DYKDDDDK Tag (14,793, Cell Signaling, Danvers, MA), anti-HA (ab9110, Abcam), anti-GAPDH antibody (AP0063, Bioworld), and the corresponding secondary antibodies, goat anti-rabbit IgG (A0208, Beyotime) or goat anti-mouse IgG (A0216, Beyotime). Immobilon Western Chemiluminescent HRP Substrate kit (Millipore) was used for color development and the bands were detected using an Amersham Imager 600 (GE Healthcare Little Chalfont, Buckinghamshire, UK).
3
Western Blot Analysis of Tagged Proteins
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Whole cell extracts were prepared by rotating the cell pellets for 2 hrs at + 4°C in five volumes of the high salt lysis buffer (50 mM Tris, 300 mM NaCl, 10% glycerol, 0.5% NP-40, 1x complete ULTRA protease inhibitors (Roche)). Proteins were resolved on 10% SDS-PAGE gels, transferred to the Immobilon-P/E PVDF membrane (Merck Millipore), and immunodetected using the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and the following antibodies: anti-FLAG M2 (Sigma, F1804, 1:1000), anti-DYKDDDDK Tag (Cell Signaling, 14793, 1:1000), anti-Myc Tag (Millipore, 05–724, 1:1000), anti-mouse-HRP (GE healthcare, NA931-1ML, 1:5000), anti-rabbit-HRP (GE healthcare, NA934-1ML, 1:5000).
4
Western Blotting for Protein Analysis
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Western blotting was performed as previously described.10 (link),27 Primary antibodies against CRIP1 (15349-1-AP, RRID: AB_2878128 ), PA200 (18799-1-AP, RRID: AB_10598020 ) and USP7 (66514-1-Ig, RRID: AB_2881877 ) were purchased from Proteintech (Rosemont, IL, USA). Additionally, anti-Ubiquitin (43124, RRID: AB_2799235 ), anti-DYKDDDDK Tag (14793, RRID: AB_2572291 ), anti-LC3B (3868, RRID: AB_2137707 ), anti-GAPDH (#5174, RRID: AB_10622025 ), anti-β-Actin and secondary antibodies (anti-rabbit (#7074, RRID: AB_2099233 ) and anti-mouse (#7072, RRID: AB_331144 ) were purchased from Cell Signaling Technology. The bands were visualized using a ChemiDoc Touch Imaging System (Bio-Rad, Singapore).
5
Regulation of Estrogen Receptor Signaling
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MG132 (S2619), tamoxifen (S1238) were obtained from Selleck (Houston, TX, USA). Control siRNA (sc-37007) and USP15 siRNA (sc-76819) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). USP15-1 and USP15-2 siRNA (S0222) were from genechem (Shanghai, China). Cell-LightTM EdU Apollo 567 In Vitro Kit (C10310-1) was obtained from RiboBio (Guangzhou, China). Annexin V-FITC/PI apoptosis detection kit (KGA107) was obtained from Keygen Company (Nanjing, China). The antibodies purchased from Cell Signaling Technology (CST, MA, USA) are listed below: anti-p21 (2947), anti-USP15 (66310), anti-ubiquitin (3936), anti-estrogen receptor α (8644), anti-phospho-Rb (8516), anti-Rb (9313), anti-Cyclin D1 (2922)(55506), anti-CDK4 (12790), anti-p27 (3686), anti-GAPDH (5174), anti-K48-ub (12805), anti-K63-ub (12930), anti-DYKDDDDK-tag (14793), anti-Myc-tag (2276). Anti-ERα (ab32063) was from Abcam (USA). Anti-USP15 (67557-1-lg) was from proteintech (Chicago, USA). Lentivirus was purchased from VigeneBio (Shandong, China) and plasmids were from Genechem (Shanghai, China). Cycloheximide (CHX) and Estrogen (E8875) were received from Sigma-Adrich (Sigma-Adrich, Louis, MO). MTS (catalog no. G111) was purchased from Promega Corporation (Madison, WI, USA). Co-IP assay kit (14311D) was purchased from Life Technologies (Carlsbad, CA).
6
Protein Detection in Whole Cell Extracts
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Whole cell extracts were prepared by rotating the cell pellets for 2 hrs at + 4°C in five volumes of the high salt lysis buffer (50 mM Tris, 300 mM NaCl, 10% glycerol, 0.5% NP-40, 1x cOmplete ULTRA protease inhibitors (Roche)). Proteins were resolved on 10% SDS-PAGE gels, transferred to the Immobilon-P/E PVDF membrane (Merck Millipore), and immunodetected using the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and the following antibodies: anti-FLAG M2 (Sigma, F1804), anti-DYKDDDDK Tag (Cell Signaling, 14793), anti-Myc Tag (Millipore, 05-724), anti-mouse-HRP (GE healthcare, NA931-1ML), anti-rabbit-HRP (GE healthcare, NA934-1ML).
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