RPMI 1640 and fetal bovine serum (FBS) were purchased from Welgene, Inc. (Daegu, South Korea). Lipofectamine™ 2000 Reagent was purchased from Invitrogen (Carlsbad, CA). D-erythro- and L-threo-SPC were purchased from Matreya (Pleasant Gap, PA). MK2206 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-AKT1 and phospo-specific antibodies to detect K8 S431 were supplied from Abcam (Cambridge, UK). Anti-RhebL1 antibody and peroxidase-labeled secondary antibodies were acquired from Santa Cruz Biotechnology. Anti-phospho-AKT Ser 473 was acquired from Cell Signaling Technology (Beverly, MA). Alexa Fluor 488 donkey anti-goat antibody and Alexa Fluor 594 chicken anti-rabbit antibody were obtained from Molecular Probes, Inc. (Eugene, OR).
Peroxidase labeled secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Peroxidase-labeled secondary antibodies are a type of laboratory reagent used in various immunoassay techniques. They are designed to detect and amplify the signal from primary antibodies bound to target antigens. The peroxidase enzyme attached to the secondary antibody can catalyze a colorimetric or chemiluminescent reaction, enabling the visualization and quantification of the target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Welgene (2 mentions)
Luminata forte hrp substrate, by Merck Group (2 mentions)
Mk 2206, by Santa Cruz Biotechnology (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Primary antibodies against α tubulin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Cyclin b1, by Merck Group (1 mentions)
Non fat dry milk, by Santa Cruz Biotechnology (1 mentions)
G box, by Syngene (1 mentions)
Dot blotter, by Carl Roth (1 mentions)
Ccd camera, by Intas (1 mentions)
Cell lytic buffer, by Merck Group (1 mentions)
Rabbit anti beclin 1, by Cell Signaling Technology (1 mentions)
Rabbit anti c ebpβ, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Rabbit anti grp78, by Cell Signaling Technology (1 mentions)
Rabbit anti pparγ, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti fas, by Santa Cruz Biotechnology (1 mentions)
12 protocols using peroxidase labeled secondary antibody
1
Regulation of AKT/mTOR Signaling Pathway
2
Noscapine Conjugate Cytoskeleton Modulation
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1 ×
105 MiaPaCa-2 cells were seeded in 12-well plates and were
treated with different concentrations of noscapine conjugates for
24 h. Soluble and insoluble tubulin fractions were collected subsequently.
The soluble tubulin fractions were collected by using 200 μLpre-warmed
lysis buffer [1 mM MgCl2, 1 mM EGTA, 80 mM Pipes-KOH (pH
6.8), 10% glycerol, 0.2% Triton X-100, and 0.1% protease inhibitor
cocktail (Sigma-Aldrich)]. Lysis buffer was removed gently and mixed
with 100 μL of 5× Laemmli’s sample buffer (0.01%
bromophenol blue, 180 mM Tris-Cl pH 6.8, 7.5% β-mercaptoethanol,
15% glycerol and 6% SDS). Samples were heated to 95 °C for 3
min. To collect the insoluble tubulin fraction in the remaining cells,
200 μL of 1× Laemmli’s sample buffer was added in
each well and collected and the samples were heated to 95 °C
for 3 min. Equal volumes of the samples were resolved using SDS-polyacrylamide
gel (10%) and transferred to the poly(vinylidene difluoride) (PVDF)
membrane. Blots were then incubated with primary antibodies against
α-tubulin (Sigma) overnight at 4 °C, and membranes were
next incubated with peroxidase-labeled secondary antibodies (Santa
Cruz Biotechnology, Texas, United States) for 1 h. Membranes were
visualized using an enhanced G-BOX (Syngene, USA).
105 MiaPaCa-2 cells were seeded in 12-well plates and were
treated with different concentrations of noscapine conjugates for
24 h. Soluble and insoluble tubulin fractions were collected subsequently.
The soluble tubulin fractions were collected by using 200 μLpre-warmed
lysis buffer [1 mM MgCl2, 1 mM EGTA, 80 mM Pipes-KOH (pH
6.8), 10% glycerol, 0.2% Triton X-100, and 0.1% protease inhibitor
cocktail (Sigma-Aldrich)]. Lysis buffer was removed gently and mixed
with 100 μL of 5× Laemmli’s sample buffer (0.01%
bromophenol blue, 180 mM Tris-Cl pH 6.8, 7.5% β-mercaptoethanol,
15% glycerol and 6% SDS). Samples were heated to 95 °C for 3
min. To collect the insoluble tubulin fraction in the remaining cells,
200 μL of 1× Laemmli’s sample buffer was added in
each well and collected and the samples were heated to 95 °C
for 3 min. Equal volumes of the samples were resolved using SDS-polyacrylamide
gel (10%) and transferred to the poly(vinylidene difluoride) (PVDF)
membrane. Blots were then incubated with primary antibodies against
α-tubulin (Sigma) overnight at 4 °C, and membranes were
next incubated with peroxidase-labeled secondary antibodies (Santa
Cruz Biotechnology, Texas, United States) for 1 h. Membranes were
visualized using an enhanced G-BOX (Syngene, USA).
3
Noscapine Analogues Induce Apoptosis in MiaPaCa-2 Cells
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MiaPaCa-2 cells were incubated
in the presence of noscapine analogues, and the total cell lysates
were obtained by using the Laemmli sample buffer. Equal volumes of
the protein lysate were resolved using SDS-polyacrylamide gel (10%)
and transferred to the PVDF membrane. The membrane was blocked for
1 h at room temperature in TBS with 0.1% Tween20 (TBST) containing
5% (w/v) nonfat dry milk (Santa Cruz Biotechnology). After 5 min of
TBST wash, the membrane was incubated with primary antibodies against
Caspase-3 (C8487), CDK1 (SAB4500050), cyclin-B1 (SAB4503501), PARP
(#9542) β-actin (Sigma) at 4 °C overnight. The blots were
then incubated with peroxidase-labeled secondary antibodies (Santa
Cruz Biotechnology) for 1 h at room temperature. The membranes were
washed with TBST and then visualized using the G-BOX (Syngene, USA).
The protein expression was normalized relative to the control gene
and actin expression.
in the presence of noscapine analogues, and the total cell lysates
were obtained by using the Laemmli sample buffer. Equal volumes of
the protein lysate were resolved using SDS-polyacrylamide gel (10%)
and transferred to the PVDF membrane. The membrane was blocked for
1 h at room temperature in TBS with 0.1% Tween20 (TBST) containing
5% (w/v) nonfat dry milk (Santa Cruz Biotechnology). After 5 min of
TBST wash, the membrane was incubated with primary antibodies against
Caspase-3 (C8487), CDK1 (SAB4500050), cyclin-B1 (SAB4503501), PARP
(#9542) β-actin (Sigma) at 4 °C overnight. The blots were
then incubated with peroxidase-labeled secondary antibodies (Santa
Cruz Biotechnology) for 1 h at room temperature. The membranes were
washed with TBST and then visualized using the G-BOX (Syngene, USA).
The protein expression was normalized relative to the control gene
and actin expression.
4
Dot Blot Analysis of Secreted Proteins
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Specific proteins in the culture supernatants were detected by dot blot. Briefly, 70 µL of the cell culture supernatant was applied on a wet nitrocellulose membrane with the help of a dot blotter (Carl Roth). After blocking membranes with 5% BSA for 1 h, primary antibody incubation (Cathepsin K—CTSK—sc-48353 and osteocalcin—OC—sc-365797; both obtained from Santa Cruz Biotechnology, Heidelberg, GER) was performed at 4 °C overnight. After incubation with the corresponding peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology, Heidelberg, GER) for 2 h, chemiluminescent signals were detected by a CCD camera (INTAS, Göttingen, GER) and quantified using the ImageJ software [17 (link),18 (link)].
5
Macrophage Protein Expression Analysis
6
Western Blot Analysis of Phospho-IκBα in Mice Tissues
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Total proteins were extracted from mice tissues with ice-cold lysis buffer (Roche Applied Sciences, Indianapolis, IN, UAS) as we described in a previous publication [17 (link)]. Equal amounts of proteins were run on 4–20% polyacrylamide gels (Mini-Protean TGX; Bio-Rad Laboratories, Hercules, CA, USA), and transferred to nitrocellulose membranes (Bio-Rad Laboratories). After blocking with 4% bovine serum albumin, membranes were incubated with the anti-phospho-IкBα (1:1000; Cell Signaling Technology, Danvers, MA, USA) or polyclonal goat anti-actin (1:5000; Sigma, St. Louis, MO, USA) antibodies. The immune complexes were detected using appropriate peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology, Dallas, USA) and chemiluminescence ECL kit (Amersham™, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Specific bands were quantified by densitometry using Alpha imager 2200 software (Genetic Technologies, Inc., Miami, FL, USA).
7
Signaling Pathway Characterization Reagents
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D-erythro- SPC and L-threo-SPC were obtained from Matreya (Pleasant Gap, PA, USA). Mouse monoclonal antibody specific for thrombospondin-1 was purchased from Abcam (Cambridge, UK). Mouse monoclonal antibodies against β-actin and vimentin were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against N-cadherin and E-cadherin, monoclonal antibody specific for ERK1+ERK2 (phosphor T202+Y204+T185+Y187), and goat polyclonal antibody against STAT3 were purchased from Abcam (Cambridge, UK). Peroxidase-labeled secondary antibodies were acquired from Santa Cruz Biotechnology. Alexa Fluor 488 goat anti-rabbit and 594 goat anti-mouse antibodies were obtained from Molecular Probes, Inc (Eugene, OR, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and DMEM-F12, RPMI 1640 and defined fetal bovine serum (FBS) were obtained from Welgene, Inc (Daegu, Korea). Lipofectamine 2000 Reagent was purchased from Invitrogen (Carlsbad, CA, USA), and SB203580, SP600125, and PD98059 were purchased from Calbiochem (La Jolla, CA, USA).
8
Quantifying 5hmC and 5mC Levels
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Total genomic DNA was extracted using the DNeasy Kit (QIAGEN), denatured (0.4M sodium hydroxide, 10mM EDTA at 100°C for 10 min), and then neutralized (6.6M cold ammonium acetate, pH 7). We applied 200 ng of DNA to a prewet Amersham Hybond‐N+ membrane (GE Healthcare Life Sciences). The membrane was blocked and incubated in primary antibody overnight (anti‐5hmC and anti‐5mC 1/200; Active Motif, Inc., Carlsbad, CA, USA), followed by the appropriate peroxidase‐labeled secondary antibody (1/5000; Santa Cruz Biotechnology). Blots were visualized using Luminata Forte HRP substrate (Millipore), and quantified using ImageJ software (NIH; https://imagej.nih.gov/ij/).
9
Immunohistochemical Analysis of Brain Markers
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Sections were rehydrated
and endogenous peroxidase blocked with 1% H2O2, then with appropriate serum together with 0.1 Triton in PBS and
probed overnight at 4 °C with the following primary antibodies:
anti-GFAP (1:300, Dako, Denmark), anti-Iba1 (1:1000; Wako, Osaka,
Japan), or anti-MBP (1:200, Santa Cruz Biotechnology, CA, USA). Sections
were incubated with peroxidase-labeled secondary antibody (1:100,
Santa Cruz Biotechnology, CA, USA) for 1 h, and staining was visualized
using diaminobenzidine and counterstained with hematoxylin. Then,
sections were dehydrated and coverslipped with DPX (VWR, Leighton
Buzzard, U.K.).
and endogenous peroxidase blocked with 1% H2O2, then with appropriate serum together with 0.1 Triton in PBS and
probed overnight at 4 °C with the following primary antibodies:
anti-GFAP (1:300, Dako, Denmark), anti-Iba1 (1:1000; Wako, Osaka,
Japan), or anti-MBP (1:200, Santa Cruz Biotechnology, CA, USA). Sections
were incubated with peroxidase-labeled secondary antibody (1:100,
Santa Cruz Biotechnology, CA, USA) for 1 h, and staining was visualized
using diaminobenzidine and counterstained with hematoxylin. Then,
sections were dehydrated and coverslipped with DPX (VWR, Leighton
Buzzard, U.K.).
10
Western Blot Analysis of Autophagy and Oxidative Stress Markers
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After treatment, the cells were collected and the protein extracted with RIPA lysis buffer. The protein concentrations were quantified by the BCA method. Protein samples (30 µg) were run on 10% gels, and then transferred to the PVDF membrane. After 1 h of blocking with the 5% non-fat milk, the membranes were incubated with the primary rabbit anti-LC3B, the rabbit anti-Beclin 1 antibody, the rabbit anti-Keap1 antibody, the rabbit anti-Nrf2 antibody, the rabbit anti-ARE antibody (1:1,000; Abcam, Cambridge, MA, USA) at 4°C overnight. Washing in TBST three times, the membranes were then incubated with a peroxidase labeled secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h. The bands were washed again, enhanced with chemiluminescence reagents and visualized with the ChemiDoc™ MP Imaging System (Bio-Rad, Berkeley, CA, USA).
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