Western blotting was performed as described [60 (link)]. Primary antibodies used were: mouse α-Myc 9E10 antibody (1:2000, Enzo Life Sciences); mouse α-FLAG M2 antibody (1:1000, Sigma-Aldrich); rabbit α-GFP TP401 antibody (1:5000, Acris Antibodies); mouse α-HA F7 (1:1000, Santa-Cruz) rat α-HA (1:750, Roche); mouse α-β-Tubulin (1:5000, Covance), mouse α-α-Tubulin (1:20,000; Sigma), mouse α-GFP (1:500; Molecular probe), mouse α-ubiquitin (1:1000; Santa Cruz) Fab α-pAbp (2.5 μg in 5 ml), α-eIF4G rabbit antibody (1 μg in 5 ml), rabbit α-Osk (1:1000) antibody was a gift from A. Ephrussi.
Rat α ha
Manufactured by Roche
Sourced in Czechia
Rat α-HA is a laboratory reagent used for the detection and purification of proteins tagged with the HA (Hemagglutinin) epitope. It functions as an antibody that specifically binds to the HA tag, enabling researchers to identify and isolate HA-tagged proteins through various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Goat α mouse hrp, by Jackson ImmunoResearch (3 mentions)
Goat α rabbit hrp, by Jackson ImmunoResearch (3 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Ibright cl1000 imaging system, by Thermo Fisher Scientific (2 mentions)
Rabbit α hltf a300 230a, by Fortis Life Sciences (2 mentions)
Mouse α ung2, by OriGene (2 mentions)
Goat α lamin b1 sc 6217, by Santa Cruz Biotechnology (2 mentions)
Goat α rat hrp, by Jackson ImmunoResearch (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Benzonase, by Merck Group (2 mentions)
Mouse α gfp, by Thermo Fisher Scientific (1 mentions)
Mouse α ha, by Santa Cruz Biotechnology (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Mouse α flag, by Merck Group (1 mentions)
Goat α rabbit hrp, by Merck Group (1 mentions)
Goat α mouse hrp, by Merck Group (1 mentions)
Mouse α gapdh, by Abcam (1 mentions)
Mouse α gfp, by Santa Cruz Biotechnology (1 mentions)
Rabbit α h3k9me3, by Abcam (1 mentions)
Rabbit α h3, by Abcam (1 mentions)
8 protocols using rat α ha
1
Comprehensive Western Blot Antibody Protocol
2
Lipophorin Visualization in Drosophila
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The following antibodies were used in this work: rabbit α-ApoLTPI and α-ApoLTP II [4 (link)], rabbit α-LpFL and rabbit α-LpII [5 (link)], rat α-HA (Roche), mouse α-HA (Santa Cruz Biotechnology) and mouse α-Myc (DSHB). Immunostaining of imaginal discs and ovaries as well as immunostaining of extracellular proteins was performed as described [13 (link)]. To examine the stability of lipophorin association to Lpr2E in vivo (Fig 7 ), wing imaginal discs were dissected in Sf-900 II SFM culture media (gibco) at 4°C and incubated in the same media for 30 minutes or 60 minutes, also at 4°C. They were subsequently fixed and processed following standard protocols. Lipids were visualized by Nile red or by oil red O stains. For Nile red, fixed imaginal discs or ovaries were incubated with 0.002% Nile red dye diluted in PBS and 0.3% triton X-100 for 60 minutes and washed for 10 minutes in the same buffer without the dye. For oil red O stain, fixed imaginal discs were incubated in a 0.5% solution of oil red O in propylene glycol at 60°C for one hour and then washed twice in 85% propylene glycol and three times in PBS, essentially as described in [56 (link)].
3
Quantification of Tetherin Incorporation into Viral Particles
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A total of 4 × 105 293T cells were plated per well of a 6-well plate, allowed to adhere overnight, and PEI transfected. Then, 1.5 μg total DNA was added to 35 μL of Optimem without serum and 6 μL of 1 μg/μL PEI before brief vortexing and incubation at room temperature for 30 min. Transfection mix was added dropwise to cells and washed with fresh complete media after 8 to 16 h. After 48 h posttransfection, supernatant was harvested and filtered through a 0.45 μM filter. For analysis of tetherin incorporation into viral particles, supernatant was concentrated by ultracentrifugation over a 20% sucrose cushion. At the time of supernatant harvest, cells were lysed in 1× RIPA buffer. Immunoblotting was performed with cat serum reactive to FIV-PPR (gift of Peggy Barr), rat α-HA (Roche), mouse anti-GFP (JL-8 clone, TaKaRa Bio), or mouse anti-alpha-tubulin (Sigma). Immunoblot band density was quantified using ImageJ.
4
SDS-PAGE and Western Blotting Protocols
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SDS-PAGE and Western blotting were performed as previously described (Scharfenberger et al., 2003 (link)). The primary antibodies as indicated in the respective figures were rat α-HA purchased from Roche, mouse α-FLAG purchased from Sigma-Aldrich, mouse α-Tub1 purchased from Sigma-Aldrich, and rabbit α-Stu1 raised against the complete Stu1 protein. Secondary antibodies were mouse α-rat-AP purchased from Sigma-Aldrich (Fig. 1 E , α-HA), goat α-mouse-HRP purchased from Sigma-Aldrich (Fig. 1 E , α-FLAG), goat α-mouse-AP purchased from Sigma-Aldrich (Fig. 2, C and D , α-tubulin), goat α-rabbit-HRP purchased from Sigma-Aldrich (Fig. 2 D , α-Stu1), and Alexa Fluor 680 goat α-rabbit purchased from Invitrogen (Fig. 1, B and C ). Fluorescence of the Alexa Fluor 680 goat α-rabbit antibody was detected by the LI-COR Biosciences system. Signal quantification was performed using the Fiji software.
5
Western Blot Analysis of DNA Repair Proteins
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Cells were lysed in Laemmli buffer supplemented with 100 mM dithiothreitol (DTT) and benzonase (1:100, Sigma-Aldrich). Following denaturation at 65°C, samples were separated by SDS-PAGE and transferred to PVDF-membranes (Millipore) by TransBlot semi-dry transfer. Membranes were blocked in 5% (w/v) milk – PBS-T (0.2% Tween-20) and incubated with primary antibody in milk-PBS-T at 4°C overnight followed by incubation with HRP-conjugated secondary antibodies at room temperature (RT). Blots were developed using chemiluminescent substrates (Thermo Scientific) and visualised using either X-ray film or an iBright CL1000 imaging system (Thermo Fisher Scientific). The following antibodies were used for immunoblotting, listed by manufacturer: Primary antibodies: Abcam: Rabbit α-SLF2 (ab122480), rabbit α-VPRBP/DCAF1 (ab202587). Bethyl Laboratories: Rabbit α-HLTF (A300-230A). GeneTex: Rabbit α-SMC6 (GTX116832), rabbit α-NSMCE1 (GTX107136). NIH AIDS Reagent Program: Mouse α-Vif (#6459; (Simon et al., 1995 (link))). Origene: Mouse α-UNG2 (TA503563). Roche: Rat α-HA (11867423001). Santa Cruz: Goat α-Lamin B1 (sc-6217). Sigma-Aldrich: Mouse α-β-actin (A5316), rabbit α-ANKRD32/SLF1 (SAB2701555). Secondary antibodies: Jackson ImmunoResearch: Goat α-mouse-HRP (115-035-146), goat α-rabbit-HRP (115-035-144), and goat α-rat-HRP (115-035-143).
6
Chromatin Immunoprecipitation and Western Blot Antibodies
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The following antibodies were used for ChIP: rabbit α-H3K9me3 (Abcam ab8898), rabbit α-H3K36me3 (Abcam ab9050), mouse α-H3K9me2 (Millipore 05-1249), rabbit α-H3 (Abcam ab1719) and mouse α-IgG (Sigma I5381).
For Western blot the following antibodies were used: mouse α-GAPDH (Abcam ab9484), rabbit α-mRFP (Apronex), rat α-HA (Roche), mouse α-GFP (Santa Cruz sc-9996), rabbit α-H3K9me3 (Abcam ab8898), mouse α-tubulin kindly provided by Pavel Draber (Institute of Molecular Genetics of the ASCR, Prague, Czech Republic), goat α-mouse-HRP (Jackson ImmunoResearch), goat α-rabbit-HRP (Jackson ImmunoResearch), goat α-rat-HRP (Santa Cruz sc-2006).
For Western blot the following antibodies were used: mouse α-GAPDH (Abcam ab9484), rabbit α-mRFP (Apronex), rat α-HA (Roche), mouse α-GFP (Santa Cruz sc-9996), rabbit α-H3K9me3 (Abcam ab8898), mouse α-tubulin kindly provided by Pavel Draber (Institute of Molecular Genetics of the ASCR, Prague, Czech Republic), goat α-mouse-HRP (Jackson ImmunoResearch), goat α-rabbit-HRP (Jackson ImmunoResearch), goat α-rat-HRP (Santa Cruz sc-2006).
7
Western Blotting of DNA Repair Proteins
8
Immunofluorescence Staining of Parasites
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Parasites growing in confluent HFFs were fixed in 4% paraformaldehyde for 10 min followed by washing in PBS three times. Fixed cells were incubated for 1 hr at room temperature in blocking buffer containing 3% BSA and 0.2% Triton X-100. The cells were incubated with the designated antibodies in blocking buffer at 4°C overnight followed by washing in PBS. The cells were then incubated with secondary antibodies coupled to Alexa 488/594/568/647 at room temperature for 1 hr. The cells were finally washed with PBS and mounted with ProLong Gold Antifade Mounting solution (Invitrogen) containing DAPI (Invitrogen) and then visualized using a Nikon Eclipse E100080i microscope. Images were captured with a Hamamatsu C4742-95 CCD camera. Nikon NIS element software was used to analyze and capture images. Primary antibody dilutions were used as followed: rabbit α-HA (Cell Signaling) 1:1,000, rat α-HA (Roche), mouse α-MYC (Cell Signaling) 1:1,000, Toxoplasma α-Centrin-1 (Kerafast company) 1:2,000, rat α-IMC3 (supplied by Dr. Marc-Jan Gubbels) 1:2,000. For the visualization of brazyzoite tissue cyst walls, FITC-conjugated Dolichos biflorus lectin (Vector Laboratories) was used at a 1:500 dilution for 1 hr at room temperature as previously described (23) .
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