Immunoblot analysis was performed as previously described [22 (link), 23 (link)]. The following antibodies were used: anti-MGMT (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-RAGE (1:1,000; Cell Signaling Technology, Tokyo, Japan), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:20,000; Trevigen, Gaithersburg, MD, USA), horseradish peroxidase-linked anti-rabbit IgG (1:20,000; GE Healthcare UK Ltd., Amersham Place, Little Chalfont, UK), and horseradish peroxidase-linked whole antibody anti-mouse IgG (1:20,000; GE Healthcare).
Horseradish peroxidase linked anti rabbit igg
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Horseradish peroxidase-linked anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is used in immunoassays and other applications that require the detection of rabbit-derived primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase linked whole antibody anti mouse igg, by GE Healthcare (6 mentions)
D5b3 monoclonal antibody 9045s, by Cell Signaling Technology (3 mentions)
A19178 200ul, by Merck Group (2 mentions)
Mouse anti dpyd a 5 monoclonal antibody sc 376712, by Santa Cruz Biotechnology (2 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase linked anti mouse igg, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti rage, by Cell Signaling Technology (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Bio-Techne (1 mentions)
Anti mgmt, by Cell Signaling Technology (1 mentions)
Ab2074, by Abcam (1 mentions)
Ecl substrate, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Quick cbb, by Fujifilm (1 mentions)
Rabbit anti gapdh, by Bio-Techne (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel, by Bio-Rad (1 mentions)
16 protocols using horseradish peroxidase linked anti rabbit igg
1
Immunoblot Analysis of MGMT and RAGE
2
Western Blotting Antibodies Validation
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Western blotting was performed as described previously.24 , 25 The antibodies used were rabbit anti‐thymidylate synthase (D5B3) monoclonal antibody (9045 S; 1:1000, Cell Signaling Technologies), rabbit anti‐LC3 polyclonal antibody (PM036, 1:1000; Medical & Biological Laboratories), mouse anti‐SQSTM1(D‐3) monoclonal antibody (sc‐28359, 1:200; Santa Cruz Biotechnology), mouse anti‐beta‐actin monoclonal antibody (A19178‐200UL, 1:20,000; Sigma‐Aldrich), horseradish peroxidase‐linked anti‐rabbit IgG (1:20,000; GE Healthcare), and horseradish peroxidase‐linked whole antibody anti‐mouse IgG (1:20,000; GE Healthcare). [Correction added on [7th December 2022], after first online publication: sc‐28,358 has been corrected to sc‐28359.]
3
Western Blot Analysis of CXCR4 Expression
4
SDS-PAGE and Immunoblot Analysis of Rice Proteins
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The salt-soluble
proteins (4 μg) of NT and RBP rice were separated by SDS-PAGE
in a 10–20% acrylamide gel (D.R.C., Tokyo, Japan) and the gel
was stained with Quick-CBB (Wako Pure Chemical Industries, Osaka,
Japan). For 1D-immunoblot analysis, the separated proteins were transferred
to a 0.2 μm BA83 Protran nitrocellulose membrane (GE Healthcare).
The membrane was incubated with 0.5% (w/v) casein-PBS blocking buffer
for 2 h at room temperature and then incubated with rabbit anti-rice
RAG2 protein antibody (diluted 1:1000 with 0.1% casein–PBS)
for 1 h at room temperature. After washing three times with 0.05%
Tween-20/PBS, the membranes were incubated with horseradish-peroxidase-linked
anti-rabbit IgG (1:2000 diluted with 0.1% casein–PBS; GE Healthcare)
for 1 h at room temperature. After three more washes with 0.05% Tween-20/PBS,
the color reaction was developed with Konica Immunostain (Konica Minolta,
Tokyo, Japan) according to the manufacturer’s protocol. The
band intensity of each sample was measured using Scion Image software,
and the significance in differences of the intensity was calculated
by Student’s t-test with Bonferroni correlation.
proteins (4 μg) of NT and RBP rice were separated by SDS-PAGE
in a 10–20% acrylamide gel (D.R.C., Tokyo, Japan) and the gel
was stained with Quick-CBB (Wako Pure Chemical Industries, Osaka,
Japan). For 1D-immunoblot analysis, the separated proteins were transferred
to a 0.2 μm BA83 Protran nitrocellulose membrane (GE Healthcare).
The membrane was incubated with 0.5% (w/v) casein-PBS blocking buffer
for 2 h at room temperature and then incubated with rabbit anti-rice
RAG2 protein antibody (diluted 1:1000 with 0.1% casein–PBS)
for 1 h at room temperature. After washing three times with 0.05%
Tween-20/PBS, the membranes were incubated with horseradish-peroxidase-linked
anti-rabbit IgG (1:2000 diluted with 0.1% casein–PBS; GE Healthcare)
for 1 h at room temperature. After three more washes with 0.05% Tween-20/PBS,
the color reaction was developed with Konica Immunostain (Konica Minolta,
Tokyo, Japan) according to the manufacturer’s protocol. The
band intensity of each sample was measured using Scion Image software,
and the significance in differences of the intensity was calculated
by Student’s t-test with Bonferroni correlation.
5
Western Blotting for Protein Expression
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Western blotting analysis was performed
as previously described.16 (link),35 (link) The antibodies used
were rabbit anti-thymidylate synthase (D5B3) monoclonal antibody (9045S,
1:1000, Cell Signaling Technologies, Massachusetts), mouse anti-DPYD
(A-5) monoclonal antibody (sc-376712, 1:1000, Santa Cruz Biotechnology,
Texas), mouse anti-β-actin monoclonal antibody (A19178-200UL,
1:20 000, Sigma-Aldrich), horseradish peroxidase-linked antirabbit
IgG (1:20 000, GE Healthcare, Connecticut), and horseradish
peroxidase-linked whole-antibody antimouse IgG (1:20 000, GE
Healthcare).
as previously described.16 (link),35 (link) The antibodies used
were rabbit anti-thymidylate synthase (D5B3) monoclonal antibody (9045S,
1:1000, Cell Signaling Technologies, Massachusetts), mouse anti-DPYD
(A-5) monoclonal antibody (sc-376712, 1:1000, Santa Cruz Biotechnology,
Texas), mouse anti-β-actin monoclonal antibody (A19178-200UL,
1:20 000, Sigma-Aldrich), horseradish peroxidase-linked antirabbit
IgG (1:20 000, GE Healthcare, Connecticut), and horseradish
peroxidase-linked whole-antibody antimouse IgG (1:20 000, GE
Healthcare).
6
Western Blot Analysis of Cellular Proteins
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Western blot analysis was performed as described previously [30 (link), 31 (link)]. The following antibodies were used: rat anti-NAMPT clone 14A.5 (1:1,000, Millipore), rabbit anti-GAPDH (1:20,000, Trevigen), mouse anti-NAPRT(B-8) (1:1,000, Santa Cruz Biotechnology), rabbit anti-POTEE antibody (1:200, Abcam), mouse monoclonal anti-beta actin clone AC-15 (1:20,000, Sigma-aldrich), horseradish peroxidase-linked anti-rat IgG (1:20,000, GE Healthcare), horseradish peroxidase-linked anti-rabbit IgG (1:20,000, GE Healthcare), and horseradish peroxidase-linked whole antibody anti-mouse IgG (1:20,000, GE Healthcare).
7
Western Blot Analysis of Islet Protein
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After 24 h of coculture, total proteins were extracted from islets using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitor. Homogenates were purified by centrifugation at 12,000 × g at 4°C for 12 min, and protein concentrations were determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific). Equal amounts of protein extract were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% Tris-HCl gels (Bio-Rad Laboratories Inc, Hercules, CA, USA). The separated proteins were then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc) and incubated for 1 h with the following primary antibodies: anti-signal transducer and activator of transcription (STAT) 3 (Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated STAT3 (pSTAT3) (Cell Signaling Technology), and anti-β actin (Sigma-Aldrich). Next, the membranes were incubated with horseradish peroxidase–linked anti-rabbit IgG (GE Healthcare, Buckinghamshire, UK) at room temperature for 1 h. The antigen–antibody complex was detected using the ECL Prime Western Blotting Detection kit (GE Healthcare).
8
Signaling Pathway Analysis of FGFR3
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Cells were treated with indicated concentrations of inhibitors in DMEM-F12 containing 10% FBS for 2 h. Subsequently, cells were lysed in 1% NP-40 lysis buffer containing protease and phosphatase inhibitors for 15 min on ice. Cells lysates were subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using primary antibodies against FGFR3 (1:1000; clone C51F2), phospho-FGFR (Tyr653/654) (1:1000), p44/42 MAPK (1:1000; clone137F5), phospho-p44/42 MAPK (Thr202/204) (1:1000), and β-Actin (1:1000; clone13E5). All primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The secondary antibody was horseradish peroxidase-linked anti-rabbit IgG (1:10,000, NA934V; GE Healthcare, Chicago, IL, USA). All blots were derived from the same experiment and processed in parallel.
9
Lipoylated Protein Analysis in Parasites
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EPI pellets were washed twice with PBS and resuspended in lysis buffer (0.5% Nonidet P-40, 500 mM NaCl, 5 mM EDTA, 1 mM DTT, 50 mM Tris-Base, 0.4% SDS, pH 7.4) at 1 × 106 parasites/μl. Samples were sonicated using 3 pulses of 30 s at 100% amplitude (Q700 sonicator, QSONICA), with 15 s breaks between to rest the tubes on the ice. Samples were centrifuged at 16,000g for 20 min and the supernatant was collected. Equivalent to 1 × 106 parasites were resolved by electrophoresis on a Mini-Protean TGX precast gel (Bio-Rad). Lipoylated proteins were detected with rabbit antilipoic acid antibody (ab 58,724, Abcam). Bound antibodies were detected with horseradish peroxidase-linked anti-rabbit IgG (GE Healthcare) and ECL Prime Western Blotting Detection Kit (GE Healthcare). The loading control was performed with mouse anti-α-tubulin (Sigma-Aldrich) and bounded antibodies were detected with horseradish peroxidase-linked anti-mouse IgG (GE Healthcare). Quantification was performed after normalization of lipoic acid signal to tubulin, using FIJI (57 (link)). Each experiment was carried out in at least three independent experiments.
10
Western Blot Analysis of DNA Repair Proteins
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Western blot analysis was performed as previously described26 , 27 , 28 , 29 with the following antibodies: mouse anti‐PARG (D8B10) monoclonal antibody (1:500; MABS61, Sigma‐Aldrich), rabbit anti‐PARP polyclonal antibody (1:1000; #9542, Cell Signaling Technologies, Danvers, MA, United States), mouse anti‐BRCA1(D‐9) monoclonal antibody (1:200; sc‐6954, Santa Cruz Biotechnology, Dallas, MA, United States), mouse anti‐poly(ADP‐ribose) monoclonal antibody (10H) (1:500; ALX‐804‐220, Enzo Life Sciences, Farmingdale, NY, United States), rabbit anti‐GAPDH antibody (1:20000; 2275‐PC‐100, Trevigen, Gaithersburg, MD, United States), horseradish peroxidase‐linked anti‐rabbit IgG (1:20000, GE Healthcare, Marlborough, MA, United States), and horseradish peroxidase‐linked whole antibody anti‐mouse IgG (1:20000, GE Healthcare).
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