Nitrocellulose membranes following transfer and blocking were incubated in primary antibody (mouse anti-His (Sigma Aldrich) 1:3000 or mouse anti-GFP (Sigma Aldrich) 1:1000) followed by incubation in a secondary coupled antibody coupled to alkaline phosphatase (Anti-Mouse IgG (H+L), AP Conjugate (Promega) 1:5000). Bands were visualised by incubation in substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT).
Mouse anti his
Manufactured by Merck Group
Sourced in United States
The Mouse anti-His is a laboratory reagent used for the detection and purification of proteins containing a histidine (His) tag. It is a monoclonal antibody that specifically binds to the His tag, which is commonly used as a fusion tag to facilitate the identification and isolation of recombinant proteins. The Mouse anti-His provides a reliable and efficient tool for protein analysis and purification in a variety of experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg hrp, by Merck Group (2 mentions)
Mouse anti gfp, by Merck Group (1 mentions)
Rabbit anti gfp, by Merck Group (1 mentions)
Protein inhibitor, by Roche (1 mentions)
Mouse anti poly adp ribose, by GeneTex (1 mentions)
Rabbit anti zikv ns3, by GeneTex (1 mentions)
Rabbit anti gfp, by Abcam (1 mentions)
Mouse anti β actin, by ZSGB-BIO (1 mentions)
Mouse anti ha, by Merck Group (1 mentions)
His 1, by Merck Group (1 mentions)
Mouse anti myc 9e10, by Developmental Studies Hybridoma Bank (1 mentions)
Rabbit anti ha, by Merck Group (1 mentions)
Rabbit anti p65, by Santa Cruz Biotechnology (1 mentions)
Mouse anti tnfr1, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti gfp, by AMS Biotechnology (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Rabbit anti tsg101, by Atlas Antibodies (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Amersham ecltm western blotting detection reagents, by GE Healthcare (1 mentions)
Semi dry blotting unit, by Thermo Fisher Scientific (1 mentions)
11 protocols using mouse anti his
1
Western Blot Protein Detection
2
Comprehensive Cell Wall Polysaccharide Profiling in P. patens
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The probes used for labeling cell wall polysaccharides in P. patens were chosen based on an earlier Comprehensive Microarray Polymer Profiling (CoMPP) analysis (Moller et al., 2007 (link)) with some additions (Table 1 ). Antibodies included anti-homogalacturonan (HG) JIM5, JIM7, LM18, LM19, LM20 (Verhertbruggen et al., 2009 (link)), anti-1-4-β-D -galactan LM5 (Jones et al., 1997 (link)), anti-1-5-α-L -arabinan LM6 (Willats et al., 1998 (link)), anti-1-3-β-D -glucan BS400-4 (Meikle et al., 1991 (link)), anti-xylan LM10 (McCartney et al., 2005 (link)), anti-xyloglucan LM15 (Marcus et al., 2008 (link)), anti-mannan BS400-4 (Pettolino et al., 2001 (link)), and anti-AGP LM2 (Smallwood et al., 1996 (link)) and JIM13 (Knox et al., 1991 (link)). CBMs used for labeling included CBM3a and CBM28 (Blake et al., 2006 (link)). Anti-extensin probes were not tested based on lack of cross-reactivity shown by CoMPP (Moller et al., 2007 (link)). Antibodies designated JIM and LM, along with CBM3A, were obtained from Plant Probes (Leeds, UK) and antibodies designated BS were obtained from Australian Biosupplies (Bundoora, VIC, Australia). CBM28 was a gift of Paul Knox (University of Leeds). Other antibodies used included Alexafluor 488-conjugated anti-mouse and anti-rat (Life Technologies, Grand Island, NY, USA) and mouse anti-His (Sigma–Aldrich, St. Louis, MO, USA).
3
Antibody Characterization for Cell Biology
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The following primary antibodies were used at a dilution of 1:1000: rabbit anti-APC2 (Cell Signaling Technology, CST12301), rabbit anti-CUL4A (Bethyl, A300-739A), rabbit anti-APC4 (Bethyl, A301-176A), rabbit anti-V5 (Abcam, ab9116), mouse anti-SUMO2/3 (Abcam, ab81371), mouse anti-SUMO2/3 (MBL-Sanbio, M114-3), rabbit anti-GFP (Sigma, 1814460), mouse anti-His (Sigma, H1029), rabbit anti-SART1 (custom-made by Eurogentec)67 (link), rabbit anti-CDC27 (Santa Cruz, sc9972), rabbit anti-CDH1, rabbit anti-Hsl1 and rabbit anti-APC11 (kind gifts from Dr. J.M. Peters, Vienna)66 (link), rabbit anti-KIF18B (Bethyl, A303-982A), and rabbit anti-Securin (Cell Signaling Technology, CST13445).
4
Comparative Analysis of GAPDH Variants
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Lf (3 μg), BSA (5 μg), wt rGAPDH (1 μg), rGAPDH (N142S) (1 μg), or rGAPDH (P295L) (1 μg) were resolved on 10% SDS-PAGE and then transferred to nitrocellulose membrane. The membrane was then blocked with 5% BSA and probed with 10 μg/ml of either wt rGAPDH, rGAPDH(N142S) or rGAPDH(P295L) for 2 h, followed by incubation with mouse anti-His (Sigma) (1:3,000) for 1 h and anti-mouse IgG HRP (Sigma) (1:8,000) for 1 h. Finally, blots were washed and developed with TMB/H2O2; the experiment was repeated three times.
5
Cell Protein Expression Analysis
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All cells were treated as indicated and lysed with lysis buffer [50 mM tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM PMSF, and 1 × protein inhibitor (Roche)]. The cell extracts were immunoblotted with the indicated antibodies to measure the level of the expressed proteins. Mouse anti-β-actin (ZSGB-Bio), rabbit anti-GFP (Abcam), rabbit anti-PARP11 (Finetest), rabbit anti-IFNAR1(Abcam), mouse anti-poly (ADP-ribose) (GeneTex), rabbit anti ZIKV NS1 (Genetex), rabbit anti-ZIKV NS3 (Genetex), mouse anti-HA, mouse anti-His, and mouse anti-Flag tag antibodies (Sigma-Aldrich) were used for detection at the appreciated dilutions.
6
Immunogold Labeling for Transmission Electron Microscopy
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Grids were equilibrated in one drop of TBST (20 mM Tris–HCl pH 7.2, 500 mM NaCl, 0.05% Tween 20, 0.1% BSA) before being transferred into a drop of 2% BSA in TBST and incubated at room temperature for 30 min. Grids were then immediately transferred into a 20 μL drop of primary antibody (mouse anti-his (Sigma Aldrich 1:10)) and incubated for 1 h. Grids were washed in a fresh drop of TBST followed by washing for 10 s in a stream of TBST. Grids were equilibrated in a drop of secondary antibody (Goat anti-mouse IgG 10 nm gold (Agar Scientific 1:50)) then incubated for 30 min in a fresh drop. Excess antibody was removed by washing in two drops of TBST before washing in a stream of ddH2O and dried.
7
Immunofluorescence and Western Blot Antibodies
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Primary antibodies used were rabbit anti‐p65 from Santa Cruz (clone C‐20, ref. sc‐372) at 1/250, mouse anti‐myc9E10 (developed by Bishop, J.M. was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa), mouse anti‐HA (Eurogentec, clone 16B12, ref. MMS‐101R), rabbit anti‐HA (Sigma, ref. H6908), rabbit anti‐GFP (Amsbio, ref. TP401), rabbit anti‐UBAP1 (Proteintech, ref. 12385‐1‐AP), mouse anti‐His (Sigma, clone HIS‐1, ref. H1029), mouse anti‐FLAG (Sigma, clone M2, ref. F1804) all at 1/1000 and mouse anti‐TNFR1 (Santa Cruz, clone H‐5, ref. sc‐8436) and rabbit anti‐TSG101 (Atlas Antibodies, ref. HPA006161) both at 1/200. Rabbit polyclonal anti‐PumA serum was obtained by repeated immunization of rabbits with purified PumA (Eurogentec) and was used at 1/1,000 for Western blot and for immunofluorescence microscopy. Purified BtpA was used to obtain chicken anti‐BtpA (Eurogentec). Anti‐EF‐Tu antibody (kind gift from R. Voulhoux) was used at 1/10,000.
Secondary antibodies used were anti‐rabbit, mouse, chicken or rat conjugated with Alexas‐488, ‐555 or ‐647 all from Jackson ImmunoResearch. When necessary, phalloidin‐568 (1/1,000) was used to label the actin cytoskeleton and DAPI nuclear dye (1/1,000) for the host cell nucleus. For Western blots, anti‐mouse or rabbit‐HRP antibodies were used at 1/5,000.
Secondary antibodies used were anti‐rabbit, mouse, chicken or rat conjugated with Alexas‐488, ‐555 or ‐647 all from Jackson ImmunoResearch. When necessary, phalloidin‐568 (1/1,000) was used to label the actin cytoskeleton and DAPI nuclear dye (1/1,000) for the host cell nucleus. For Western blots, anti‐mouse or rabbit‐HRP antibodies were used at 1/5,000.
8
Purification and Characterization of Mutant GAPDH Enzymes
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Both mutant forms of the protein were expressed and purified as described previously for wt rGAPDH (Boradia et al., 2016 (link)). Purification was confirmed by western blotting, detection was done after incubation with Mouse anti-His (Sigma) (1:3,000) for 1 h followed by incubation with anti-Mouse IgG HRP (Sigma) (1:8,000) for 1 h. The enzyme activity of wt rGAPDH, rGAPDH(N142S), and rGAPDH(P295L) purified from cytosol fraction was studied by measuring the increase in the absorbance at 340 nm due to oxidative reduction of NAD+ to NADH. The reaction mixture containing 200 μl of enzyme assay buffer (50 mM HEPES, 10 mM sodium arsenate, and 5 mM EDTA, pH 8.5), 1 mM NAD+ and 2 mM glyceraldehyde-3-phosphate (G-3-P) was added to wells containing 100 ng of each purified enzyme at 25°C. Enzyme activity was measured at 340 nm for 5 min on a Tecan Infinity M200 multimode microplate reader. Negative controls were set up without the specific substrate G-3-P and their values were subtracted from the final absorbance. The experiment was repeated thrice in triplicates. The enzymatic activity of wt rGAPDH was taken as 100% and data was plotted as % residual activity ± SD. Statistical analysis was done using Student's t-test.
9
Western Blot Analysis of Protein Samples
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Samples analyzed with SDS-PAGE (12.5%) were transferred onto a 0.45 μm of polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, United States) using a semi-dry blotting unit (Fisher Scientific, Hampton, NH, United States) at 450 mA for 40 min. The membranes were treated as described before (Loto et al., 2017 (link)). Primary antibodies used were rabbit anti-LotP, rabbit anti-FLAG (1:5,000, Sigma-Aldrich Corp., St. Louis, MO, United States) and mouse anti-His (1:3,000, Sigma-Aldrich). The secondary antibodies conjugated to horseradish peroxidase for chemiluminescence detection were anti-rabbit (1:20,000, Sigma-Aldrich) and anti-mouse (1:10,000, Sigma-Aldrich). Horseradish peroxidase (HRP) activity was detected using Amersham ECLTM Western Blotting Detection Reagents (GE Healthcare, Pittsburgh, PA, United States). The bands in the membrane were visualized using the automatic imager FluorChem R (ProteinSimple, San Jose, CA, United States), and the band intensity was quantified by ImageJ software (Schneider et al., 2012 (link)).
10
Western Blot Protein Detection Protocol
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The protein samples were applied to 10–15 % acrylamide SDS gels and transferred to nitrocellulose membranes (Merck Millipore, Darmstadt, Germany) by electroblotting. After blocking in 5 % non-fat milk in PBS containing 0.05 % Tween 20, membranes were probed with various primary antibodies: polyclonal rabbit anti-human Prdx1 antibody (Abcam), mouse anti-His (Sigma-Aldrich), followed by detection with anti-rabbit-IgG-horse-radish peroxidase conjugate (Sigma-Aldrich) or anti-mouse IgG horseradish peroxidase conjugate (Stratagene, Agilent Technologies, Santa Clara, CA. USA). Western blots were developed using advanced chemiluminescence (ECL) reagents (GE Healthcare, Amersham, UK) and exposed to autoradiography using Hyper-film (GE Healthcare).
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