Anti-FLAG M2 monoclonal, anti-actin, and anti-α-tubulin antibodies were obtained from Sigma-Aldrich. The anti-HA high-affinity antibody was obtained from Roche Applied Science. Anti-GFP rabbit polyclonal antibody and secondary antibodies conjugated with Alexa Fluor 350, 488, and 594 were obtained from Invitrogen. Anti-GFP mouse monoclonal and polyclonal antibodies were purchased from Clontech. Anti-Cdc20, anti-Cdc27, and anti-Ub (P4D1) antibodies were obtained from Santa Cruz Biotechnology. Antiphosphohistone H3 antibody was obtained from Millipore.
Anti ha high affinity antibody
Manufactured by Roche
Sourced in Germany, United States
The Anti-HA high-affinity antibody is a laboratory reagent designed for the detection and purification of proteins tagged with the HA (Hemagglutinin) epitope. This antibody exhibits a high binding affinity to the HA tag, enabling efficient capture and identification of HA-tagged proteins in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Monoclonal anti flag m2, by Merck Group (2 mentions)
Rabbit anti gfp polyclonal antibody, by Thermo Fisher Scientific (2 mentions)
Rat anti ha, by Roche (2 mentions)
Anti actin, by Merck Group (1 mentions)
Anti ub p4d1, by Santa Cruz Biotechnology (1 mentions)
Anti phospho histone h3 antibody, by Merck Group (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Dykddddk tag antibody, by Cell Signaling Technology (1 mentions)
Ezview red anti ha affinity gel beads, by Merck Group (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Hybond p, by Cytiva (1 mentions)
Anti gfp antibody a11122, by Thermo Fisher Scientific (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Roche (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Monoclonal mouse anti gfp, by Takara Bio (1 mentions)
Anti dsred polyclonal antibody, by Takara Bio (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Sc 9104, by Santa Cruz Biotechnology (1 mentions)
12 protocols using anti ha high affinity antibody
1
Antibody Sourcing for Protein Analysis
2
Purification and Detection of NT5C2 Proteins
3
Constructing Designer TALEs for Xanthomonas
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Designer TALEs (dTALEs) containing the repeat arrays elaborated in Fig 3A S4 Fig
4
Protein Extraction and Western Blot Analysis
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Protein extracts were prepared as previously described [50 (link)]. In brief, cells from 5- to 10-ml mid-log cultures were harvested and resuspended in 1 ml of 20% trichloroacetic acid (TCA). The supernatant was removed after centrifugation, and the pellet was resuspended in 100 μl of 20% TCA. Cells were broken using a FastPrep FP120 cell disrupter (BIO 101 ThermoSavant) and the lysate was recovered. Lysates were centrifuged at 1000×g for 3 min, and the pellet was thoroughly resuspended in 100 μl of 2×Laemmli buffer and 50 μl of 2 M Tris base. After boiling for 5 min, 10–20 μl were loaded in the gels. Proteins were resolved by 10% SDS-PAGE, transferred to Hybond-P (Amersham Bioscience) membranes and probed with anti-HA High Affinity antibody (3F10, Roche). Horseradish peroxidase-conjugated anti-rat antibody (NA935V, GE Healthcare) was used as secondary antibody. Detection of proteins was performed using the Luminata Forte Western HRP substrate (Merck Millipore).
5
Co-immunoprecipitation of Pku70-HA, Ssb3-YFP, and Rad11-YFP
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5.108 cells were harvested, washed in cold water and resuspended in 400 μl of EB buffer (50 mM HEPES High salt, 50 mM KOAc pH 7.5, 5 mM EGTA, 1% triton X-100, 1 mM PMSF, and protease inhibitors). Cell lysis was performed with a Precellys homogenizer. The lysate was treated with 250 mU/µl of benzonase for 30 min. After centrifugation, the supernatant was recovered and an aliquot of 50 µl was saved as the INPUT control. 2 µl of anti-GFP antibody (A11122 from Life Technologies, dilution 1:150) were added to 300 µl of protein extract and incubated for 1 h 30 min at 4 °C on a wheel. Then, 20 µl of Protein G Dynabeads (Invitrogen, 10003D), prewashed in PBS, were added and then incubated at 4 °C overnight. Beads were then washed twice 10 min in EB buffer before migration on acrylamide gel for analysis by Western blot. Pku70-HA, and Ssb3-YFP and Rad11-YFP were detected using anti-HA high-affinity antibody (Roche, 11867423001, 1:500) and using anti-GFP antibody (Roche, 11814460001, 1:1000), respectively. The Supplementary Fig. 5 shows that Pku70-HA was slightly interacting in an unspecific way with anti-GFP antibody. However, the intensity of Pku70-HA in the IP fraction was highly increased in cells expressing SSb3-YFP or Rad11-YFP, showing that most interactions with Pku70-HA are specific.
6
Generating Arabidopsis Lines Expressing HaRxL21
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35S::HaRxL21 lines (RxL21a/b) are described in Fabro et al. [16 (link)]. To generate 35S::HA::HaRxL21 lines in Arabidopsis, RxL21 and RxL21ΔEAR were cloned into pEarleygate201 [97 (link)] and transformed into Arabidopsis ecotype Col-4 using floral dipping {Clough:1998vw}. Independent transformants were selected on BASTA (Bayer CropScience, Wolfenbüttel, Germany) until homozygous. Western Blotting was performed on 14 day old seedlings to determine protein expression using anti-HA high affinity antibody (Roche, Penzberg, Germany). Primer sequences for RxL21 and RxL21 deletion variants are in S9 Table . To generate estradiol inducible lines, RxL21 and RxL21ΔEAR were cloned into the pER8 plasmid {Zuo:2000} and transformed into Arabidopsis (Col-0 background) via floral dip. Independent homozygous lines were selected on hygromycin (Invitrogen, Carlsbad, US).
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Detailed Protocols for Complex I Subunit Analysis
8
Antibody Production and Characterization
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Anti-CAMDI antibody was produced by immunizing a rabbit with synthetic peptide in previously described [13 (link)]. Anti-KIBRA antibody was produced by immunizing a rabbit with synthetic peptide (RRRLEKDLQAARDTQSK ). Anti-FLAG M2 monoclonal and anti-α-tubulin antibody were obtained from Sigma-Aldrich. Anti-HA high affinity antibody obtained from Roche. Anti-GFP rabbit polyclonal antibody and secondary antibodies conjugated with Alexa Fluor 350, 488, 594 were obtained from Invitrogen. Anti-GFP mouse monoclonal and Anti-DsRed polyclonal antibody were purchased from Clontech. Anti-surface GluA2 antibody was purchased from Chemicon.
9
Subcellular Localization of RAI1 Mutants
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To study the expression of the mutant protein, Neuro-2a cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and the plasmid pALTER-MAX RAI1-HA wild type or RAI1-HA c.3440G > A. All transfections were performed according to the manufacturer’s protocol. Western blot analysis was performed as previously described [26 (link)]. Immunodetection was performed using rat anti-HA (1:7000, Roche, Branford, CT, USA) and rabbit anti-β-tubulin (1:1000, sc-9104 Santa Cruz, Dallas, TX, USA). Results were visualized by chemiluminescence. For immunofluorescence, cells were fixed 24 h after transfection with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100 in PBS. Subcellular localization of RAI1-HA wild type and mutant forms were detected using the anti-HA high affinity antibody (1:1000, clone 3F10, Roche, Branford, CT, USA). Secondary antibody conjugated to Alexa Fluor 488 (1:1000) was used. Cells were stained with 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) and mounted with a Dako fluorescent mounting medium.
10
Arabidopsis Protein Degradation Assay
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Seven-day-old seedlings from the Arabidopsis lines overexpressing ZmOST1-HA, ZmOST1[ck2A]-HA, and ZmOST1[ck2E]-HA were ground in liquid nitrogen and resuspended in degradation buffer (25 mm Tris-HCl pH 7.5, 10 mm NaCl, 10 mm MgCl 2 , 5 mm DTT, 4 mm PMSF, 10 mm ATP). Extracts (40 mg) were incubated at room temperature with or without 40 mM MG132 (Sigma) for the indicated times. Reactions were stopped by adding protein gel-loading buffer. Protein levels over the time course were assessed by Western blot using anti-HA high-affinity antibody (Roche).
Cell-free degradation assays were performed three times.
Cell-free degradation assays were performed three times.
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