Proteins separated by 1-D and 2-DE gel electrophoresis were transferred onto nitrocellulose membranes (0.2 mm). Immediately after WB, the membranes were fixed in 7% acetic acid (v/v) and 10% methanol (v/v) for 15 min, subsequently rinsed with H2O, and stained with 1 mM RuBP (SunaTech Inc.; Suzhou, P. R. China) in 1% phosphoric acid and 30% ethanol for 15 min. Membranes were then rinsed with H2O prior to the acquisition of the total proteins by “ImageQuant LAS4010” (GE HealthCare). After acquisition, membranes were blocked with 3% low fat dried milk, 0.2% (v/v) Tween 20 in PBS for 1 h at room temperature. Subsequently, membranes were incubated 2 h at room temperature with the primary antibody for acetylated-lysine (mouse monoclonal; 1:1,000 dilution, Cell Signaling Technology Inc., MA, USA). HRP-conjugated goat antimouse (1:10,000 dilution; PerkinElmer, MA, USA) was used as a secondary antibody. Immunoblots were developed using the ECL detection system (PerkinElmer, MA, USA). The chemiluminescent images were acquired by “ImageQuant LAS4010”.
Hrp conjugated goat antimouse
Manufactured by PerkinElmer
Sourced in United States
HRP-conjugated goat antimouse is a laboratory reagent that consists of horseradish peroxidase (HRP) conjugated to goat-derived antibodies specific to mouse immunoglobulins. This reagent is commonly used in various immunoassay techniques, such as Western blotting and enzyme-linked immunosorbent assays (ELISAs), to detect and quantify the presence of mouse proteins or antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4010, by GE Healthcare (1 mentions)
Ecl detection system, by PerkinElmer (1 mentions)
Dfc365 fx camera, by Leica (1 mentions)
Opal 690, by Akoya Biosciences (1 mentions)
Cy3 streptavidin, by Jackson ImmunoResearch (1 mentions)
Biotinylated donkey anti rat, by Jackson ImmunoResearch (1 mentions)
Mouse anti perk, by Merck Group (1 mentions)
2 protocols using hrp conjugated goat antimouse
1
Acetylated Protein Detection by Western Blot
2
Quantitative Analysis of pERK in 4T1 Liver Metastases
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Following 21 days of 4T1 breast cancer cell injection, livers were collected, fixed in 4% paraformaldehyde, and embedded in paraffin blocks and 4-μm sections were made. Slides were deparaffinized, and antigen retrieval was done using citric acid pH 6. Blocking for unspecific binding was done with 20% normal horse serum (NHS) and 0.1% Triton in PBS. Rat anti-CD45 (Bio-Rad; #MCA1031G) and mouse anti-pERK 1:100 (Sigma; #M8159) were diluted in 2% NHS and 0.1% Triton and were incubated overnight. Slides were then incubated with biotinylated donkey anti-rat 1:100 (Jackson ImmunoResearch; #712-065-153) and HRP-conjugated goat anti-mouse 1:100 (PerkinElmer; #NEF822001EA) diluted 2% NHS for 1.5 hours. Slides were then incubated with 1:500 OPAL 690 (Akoya Biosciences; #FP1497001KT) and streptavidin Cy3 (016-160-084; Jackson ImmunoResearch). Slides were imaged with a Leica Mi8 microscope equipped with a motorized stage and a Leica DFC365 FX camera. Single ×20 magnification images were tiled to receive a full scan of the tumor section. The quantification in the liver sections stained with pERK was done by ImageJ.
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