Purified recombinant proteins were mixed with 5 × SDS reducing loading buffer and boiled for 8 min at 100 °C and then resolved on 10% to 12% SDS-PAGE followed by Coomassie brilliant blue staining. For Western blot analysis, purified recombinant proteins, protein pulldowns, and eluted proteins were separated by 10% to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore) and block with 5% skimmed milk in Tris-buffered saline-Tween 20 for 2 h at room temperature (RT). After washing with Tris-buffered saline-Tween 20 three times, the polyvinylidene difluoride membranes were incubated with primary antibodies overnight at 4 °C and then with a horseradish peroxidase-labeled goat anti-mouse or anti-rabbit IgG antibody (SouthernBiotech) for 1.5 h at RT. The fluorescence signals were visualized using an enhanced chemiluminescence kit (New cell & Molecular Biotech) and analyzed using a ChemiDoc MP imaging system (Bio-Rad). The following primary antibodies were used: mouse anti-His antibody (ABclonal), mouse anti-HA antibody (ABclonal), rabbit anti-GST antibody (Proteintech), rabbit anti-Flag antibody (Proteintech), mouse anti-PfGAMA-Tr3 antisera, rabbit anti-ANK1-F2 antisera, and rabbit anti-band 3-P5 antisera.
Mouse anti ha antibody
Manufactured by ABclonal
Sourced in United States, China
The Mouse anti-HA antibody is a primary antibody that specifically recognizes the hemagglutinin (HA) tag, a commonly used epitope tag for protein expression and detection. This antibody can be used to identify and immunoprecipitate HA-tagged proteins in various applications, such as Western blotting, immunoprecipitation, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Rabbit anti flag antibody, by Proteintech (1 mentions)
Rabbit anti gst antibody, by Proteintech (1 mentions)
Mouse anti his antibody, by ABclonal (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Anti flag antibody, by ABclonal (1 mentions)
Horseradish peroxidase conjugated goat anti mouse antibody, by Bioss Antibodies (1 mentions)
Magnetic bead, by Merck Group (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Rabbit anti hur antibody, by Merck Group (1 mentions)
Mouse anti ago2 antibody, by Merck Group (1 mentions)
Rabbit anti flag antibody, by Merck Group (1 mentions)
Clarity western ecl, by Bio-Rad (1 mentions)
Dynabeads protein g beads, by Thermo Fisher Scientific (1 mentions)
4 protocols using mouse anti ha antibody
1
SDS-PAGE and Western Blot of Recombinant Proteins
2
Immunodetection of HA and FLAG Proteins
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Mouse anti-HA antibody and anti-FLAG antibody were purchased from Abclonal Technology. Horseradish peroxidase-conjugated goat anti-mouse antibody was purchased from Bioss.
3
RIP Assay for Protein-RNA Interactions
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RIP assays were conducted using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, USA). Briefly, MGC-803 cells were lysed in RNA lysis buffer with protease and RNase inhibitors. The cell lysates were incubated with magnetic beads conjugated with 5 μg negative control lgG (Cat# CS200621, Cat# PP64B, Millipore, Billerica, USA), positive control anti-snRNP70 antibody (Cat# CS203216, Millipore, Billerica, USA), mouse anti-AGO2 antibody (Cat# 03-110, RRID: AB_10615426, Millipore, Billerica, USA), rabbit anti-HuR antibody (Cat# 03-102, RRID: AB_11211202, Millipore, Billerica, USA) or Mouse anti-HA antibody (Cat# AE008, RRID: AB_2770404, ABclonal, USA) and rotated at 4 °C overnight. The beads were then washed and digested with proteinase K buffer at 55 °C for 30 min to remove proteins. The immunoprecipitated RNA was extracted and purified for RT-qPCR analysis.
4
Co-immunoprecipitation of RNS1 and Fus3
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The master plasmid pPK2-sur-Ptef-FLAG or pPK2-bar-Ptef-HA was used for producing a protein fused with FLAG or HA, respectively (26 (link)). To assay the in vivo interaction between RNS1 and Fus3, an RNS1-FLAG/Fus3-HA strain expressing the fusion protein RNS1::FLAG and Fus3::HA was constructed. The coding sequence of RNS1 was inserted into pPK2-sur-Ptef-FLAG, and the resulting plasmid was then transformed into the WT strain to produce the WT-RNS1-FLAG strain. The coding sequencing of Fus3 was inserted into the plasmid pPK2-bar-Ptef-HA to produce pPK2-sur-Ptef-Fus3-HA, which was transformed into the WT-RNS1-FLAG strain to produce the RNS1-FLAG/Fus3-HA strain.
Extraction of total fungal proteins and Co-IP analysis were performed as described previously (26 (link)). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Meilune, Dalian, China). The mouse anti-HA antibody was purchased from Abclonal (Hangzhou, China), and the rabbit anti-FLAG antibody was purchased from Sigma-Aldrich (MO, USA). Dynabeads protein G beads were purchased from Invitrogen (CA, USA). Detection of a target protein was conducted using Western blot analysis. All blots were imaged by a chemiluminescence detection system (Clarity Western ECL; Bio-Rad). All Co-IP assays were repeated three times.
Extraction of total fungal proteins and Co-IP analysis were performed as described previously (26 (link)). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Meilune, Dalian, China). The mouse anti-HA antibody was purchased from Abclonal (Hangzhou, China), and the rabbit anti-FLAG antibody was purchased from Sigma-Aldrich (MO, USA). Dynabeads protein G beads were purchased from Invitrogen (CA, USA). Detection of a target protein was conducted using Western blot analysis. All blots were imaged by a chemiluminescence detection system (Clarity Western ECL; Bio-Rad). All Co-IP assays were repeated three times.
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