Equal amounts of protein sample (30 µg) were separated through 7–12.5% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were activated with methanol and blocked with 1% bovine serum albumin (BSA) in TBS-T (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20) at room temperature, then incubated with primary antibodies (Table 1 ) at 4 °C. After overnight incubation, the membranes were washed with TBS-T and incubated with secondary antibodies for 2 h at room temperature. The secondary antibodies were used anti-mouse Ig G (cat. no. 7076S; Cell Signaling Technology, Inc., Beverly, MA, USA), anti-rabbit Ig G (cat. no. 7074S; Cell Signaling Technology, Inc., Beverly, MA, USA) and donkey anti-goat Ig G (cat. no. A50-101p; Bethyl Laboratories, Inc., Montgomery, TX, USA) diluted 1: 10,000 in TBS-T. The enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to detect immunoreactivity. Densitometric analysis was conducted through triplicate experiments and analyzed using Multi-Gauge software (version 3.0; Fujifilm Life Science, Tokyo, Japan).
Donkey anti goat igg
Manufactured by Cell Signaling Technology
Sourced in United States
Donkey anti-goat IgG is a secondary antibody used in various immunoassays and immunodetection techniques. It is raised in donkeys and specifically binds to the Fc region of goat immunoglobulin G (IgG) molecules.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence ecl kit, by Thermo Fisher Scientific (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Phenylmethylsulfonyl fluoride, by Thermo Fisher Scientific (1 mentions)
Nonfat milk powder, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Nupage 10 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pvdf membranes 0.45 m, by Merck Group (1 mentions)
Non fat milk powder, by BD (1 mentions)
Imagequant las 4000, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Enhanced chemiluminescence western blot kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Multi gauge software version 3, by Fujifilm (1 mentions)
Bioanalytical imaging system, by Azure Biosystems (1 mentions)
3 protocols using donkey anti goat igg
1
Western Blot Protein Analysis Protocol
2
Immunoblotting Analysis of ALDH1A1 and CXCR4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in 6-well plate (Corning), washed once with PBS (pH 7.4), and scraped using scraper (Fisherbrand). The suspension was lysed with 200 μl of lysing buffer supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Thermo scientific) on ice for 60 min. Protein fractions were collected by centrifugation at 15,000 rpm at 4 °C for 10 min. Sample loading was normalized according to BCA (Bicinchoninic acid) relative protein quantification (Solarbio). Proteins separated following a NuPAGE 10% Bis-Tris Gel (Thermo), wet electrophoretic transfer was used to transfer the proteins to polyvinylidene difluoride (PVDF) membranes (0.45 µm; Millipore, Bedford, MA). The membranes were blocked with 5% non-fat milk powder (BD Bioscience) in Tris-buffered saline with 0.1% Tween (TBST) for 1 hour at room temperature and then incubated with ALDH1A1 antibody (rabbit mAb, Cell Signal Technology) or CXCR4 antibody (goat mAb, Abcam) overnight at 4 °C, followed by horse-radish-peroxidase conjugated goat anti-rabbit IgG or donkey anti-goat IgG (Cell Signal Technology) respectively for 1 hour at RT in 0.5% non-fat milk powder with TBST. Visualization was performed using Image Quant LAS 4000 with an Enhanced Chemiluminescence Kit (Thermo Pierce, Waltham, MA, USA).
3
Protein Separation and Quantification by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE gel (7.5-12.5%) was used to separate proteins (30 µg per lane). The proteins were then transferred to PVDF membranes (EMD Millipore). Membranes were washed with methanol, and then blocked for 2 h with TBS-T [10 mm Tris-HCl, 150 mm NaCl (pH 7.5) and 0.1% Tween-20] containing 1% BSA. The membrane were then incubated at 4°C overnight (≥12 h) with primary antibodies. After washing 2 times with TBS-T for 15 min each time, the membranes were incubated at room temperature for a further 2 h with the secondary antibodies (all 1:10,000). The secondary antibodies used were HRP-conjugated anti-rabbit IgG (cat. no. 7074S, Cell Signaling Technology, Inc.), donkey anti-goat IgG (cat. no. A50-101p, Bethyl Laboratories, Inc.) and anti-mouse IgG (cat. no. 7076S, Cell Signaling Technology, Inc.). The primary antibodies used are listed in Table I . Color development was performed using an enhanced chemiluminescence western blot kit (Thermo Fisher Scientific, Inc.). The bioanalytical imaging system (Azure Biosystems) was used to detect the protein bands. Multi-Gauge software, version 3.0 (Fujifilm Life Science) was used to analyze the density of these protein bands. Each density of these protein bands was normalized to GAPDH.
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!