For western blotting detection, epimastigotes were lysed with 1 mL RIPA buffer supplemented with 1 mM phenylmethanesulfonyl fluoride and 5 mM leupeptin, for 30 min at 4 °C. Then, homogenates were centrifuged at 16,000 rpm for 10 min. Aliquots of the supernatants (containing 100 µg total protein) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA), which were blocked with 5% milk in PBS plus 0.1% (w/v) Tween 20, probed overnight at 4 °C with the primary mouse anti-GAPDH antibody (1:500, Sigma-Aldrich), and detected using horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA). The loading control was probed with a primary rabbit anti-tubulin antibody (1:500, Sigma-Aldrich) and detected using an HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000, Santa Cruz Biotechnology). Luminescence was detected using an ImageQuant LAS 4000 digital imaging system (GE Healthcare Life Sciences, Amersham, UK) after the reaction with LuminataTM Forte Western HRP Substrate (Millipore, Billerica, MA, USA). Densitometric analysis was performed using ImageJ software version 1.50i (NIH Image, Bethesda, MD, USA) with background correction.
Horseradish peroxidase hrp conjugated anti mouse igg secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody is a laboratory reagent used in various immunoassay techniques. It contains an anti-mouse IgG antibody covalently linked to the enzyme horseradish peroxidase. This allows for the detection and quantification of mouse immunoglobulin G (IgG) proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Las 4000, by Cytiva (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bca kit, by Merck Group (1 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
3 protocols using horseradish peroxidase hrp conjugated anti mouse igg secondary antibody
1
Western Blot Analysis of GAPDH and Tubulin
2
Western Blot Analysis of MDR1 and GAPDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were washed with Tris-buffered saline (TBS). RIPA buffer was added with constant agitation for 30 min, then centrifuged for 15 min at 4°C at 12,000 rpm and the supernatant was transferred to new tubes for subsequent experiments. The protein concentrations were measured using Nanodrop2000 (Thermo Fisher Scientific, San Jose, CA, USA). Proteins (20 mg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% dried skimmed milk powder in TBS, the membranes were incubated with specific primary antibodies against MDR1, (1: 2000 dilution) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and GAPDH (1: 2000 dilution) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. The membrane was washed five times for 25 min with TBS and Tween-20 (TBST), followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1: 2000 dilution) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The protein signals were detected using Pierce™ ECL Western blot substrate (Thermo Fisher Scientific, San Jose, CA, USA).
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cultured cells were collected and lysed using RIPA buffer (Sigma-Aldrich) according to the protocol of manufacture. After total protein quantification by BCA kit (Sigma-Aldrich), an equal quantity 30μg protein each well was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretic transferred onto PVDF membrane (Millipore, MA, USA). After blocking with 5% non-fat dry milk in Tris-buffered saline with Tween-20 (TBS-T), the membranes were probed with primary anti-CREB1 antibody (1:1000 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Bax(1:1000 dilution; Santa Cruz Biotechnology Inc), anti-Bcl-2(1:1000 dilution; Santa Cruz Biotechnology Inc), anti-CYP19A1(1:1000 dilution; Santa Cruz Biotechnology Inc) and anti-GAPDH antibody (1:3000 dilution; Santa Cruz Biotechnology Inc) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:5000 dilution; Santa Cruz Biotechnology Inc) at 37 °C for 2 h. GAPDH was used as an internal control. The protein bands were visualized using an ECL detection reagent on Bio-Rad ChemiDoc MP (Bio-Rad,USA).
The relative protein intensities were analyzed using Gel-pro Analyzer® software (Media Cybernetics, Rockville, MD, USA).
The relative protein intensities were analyzed using Gel-pro Analyzer® software (Media Cybernetics, Rockville, MD, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!