Proteins with significant ubiquitination changes were verified with Western blotting. Standard Western blotting was preformed using primary antibodies against chicken CDK1 (dilution 1:1000, Abcam, Cambridge, MA, USA), chicken ETV6 (1:2000, MyBioSource, Inc., San Diego, CA, USA), chicken IL-18 (1:500, Abcam), PRKCB (1:500, LifeSpan BioSciences, Inc, Seattle, WA, USA), ETS1 (1:300, LifeSpan BioSciences), and β-actin (1:5000, Abcam). HRP-conjugated goat anti-rabbit IgG secondary antibodies (1:5000, OriGene, Rockville, MD, USA) were used for ECL imaging.
Hrp conjugated goat anti rabbit igg secondary antibody
Manufactured by OriGene
Sourced in United States
HRP-conjugated goat anti-rabbit IgG secondary antibodies are laboratory reagents used in immunoassay techniques. They are designed to detect and bind to rabbit primary antibodies, with a horseradish peroxidase (HRP) enzyme label attached for signal amplification.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Protease inhibition cocktail, by Roche (1 mentions)
M iggκ bp hrp sc 516102, by Santa Cruz Biotechnology (1 mentions)
Ecl detection kit, by Bio-Rad (1 mentions)
Gapdh antibody sc 47724, by Santa Cruz Biotechnology (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Fluoromax 4 fluorescence spectrophotometer, by Horiba (1 mentions)
4 protocols using hrp conjugated goat anti rabbit igg secondary antibody
1
Western Blot Analysis of Ubiquitinated Proteins
2
SDS-PAGE Immunoblot Analysis of Splenic Proteins
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Briefly, total proteins of splenic immune cells were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad; Cat. 1620177), which were blocked with skimmed milk and incubated with primary antibodies generated from immunized rabbits overnight at 4 °C, followed by incubation with HRP-conjugated goat anti-rabbit IgG secondary antibodies (Origene; Cat. ZB-2301). A list of antibodies for Western blotting is available in the Supporting Information (Supplementary Table 1 ). Protein bands were quantified using ImageJ software. All experiments were repeated at least three times in triplicate for each sample.
3
Protein Expression Analysis in Cardiac Tissue
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Left ventricular myocardial biopsies or cells were lysed in RIPA protein lysis buffer containing PMSF and protease inhibition cocktail (Roche Diagnostics GmbH, Mannheim, Germany) using the Bead Ruptor 4 Mini Homogenizer (Omni International). Protein concentration was determined with a BCA protein assay kit (Thermo Scientific, Rockford, USA). Protein extracts were separated on a polyacrylamide gel and subsequently transferred to a PVDF membrane. The membrane was blocked in 5% non‐fat dry milk/TBS‐T buffer followed by overnight incubation at 4°C with primary antibody. After rinsing, the membrane was incubated with HRP conjugated secondary antibody and detected by use of the ECL detection kit (Bio‐Rad). Densitometric quantification of protein bands was performed with the Chemidoc Touch Imaging System (Bio‐Rad). Following antibodies were used: anti‐SERPINA3 rabbit monoclonal antibody (ab205197, Abcam, Cambridge), Akt antibody (#9272) (Cell Signaling Technologies, Massachusetts), and HRP conjugated goat anti‐rabbit IgG secondary antibody (TA1300233, Origene). GAPDH was used to normalize for different protein content using GAPDH antibody (sc‐47724) and mIgG κ BP‐HRP (sc‐516102) (Santa Cruz Biotechnology, Inc, Dallas, Texas).
4
Characterization of UL36(480) Hydrolysis Specificity
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SUMO1-GST, SUMO2-GST, SUMO3-GST [47 ,48 ], di-SUMO2(K11), or di-SUMO3(K11) and various rhodamine-conjugated ubiquitin-like proteins (Boston Biochem) were used as substrates for the characterization of hydrolysis specificity of 1 μM UL36(480). The reactions were performed as described above using SUMO substrate, and hydrolysis products were identified by Western blotting using primary antibody against SUMO1 (Cat#: ab5316) or SUMO2/3 (Cat#: ab3742) (dilution 1:2000, Abcam), and HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000, OriGene) was used for ECL imaging. Hydrolysis specificity of UL36(480) on rhodamine-conjugated ubiquitin-like proteins was assessed in reactions containing 0.1μM of FAT10-Rhodamine, NEDD8-Rhodamine, UFM1-Rhodamine, or ISG15-Rhodamine substrates (Boston Biochem), respectively, and 1 μM UL36(480) in a final volume of 100 μL 1× reaction buffer at 37 °C. The fluorescence intensity of rhodamine group hydrolyzed from the reactions was monitored on a FluoroMax 4 fluorescence spectrophotometer (HORIBA Scientific, Edison, NJ, USA) with excitation and emission wavelengths of 570 and 590 nm, respectively.
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