The largest database of trusted experimental protocols

Gradient tris glycine precast gel

Manufactured by Thermo Fisher Scientific
Sourced in United States

4–20% gradient Tris-glycine precast gels are a laboratory equipment product designed for protein separation and analysis. These gels feature a gradient of 4% to 20% polyacrylamide concentration, allowing for the effective separation of a wide range of protein molecular weights. The Tris-glycine buffer system provides a standard and well-established method for protein electrophoresis.

Automatically generated - may contain errors

8 protocols using gradient tris glycine precast gel

1

Western Blot Analysis of KEAP1 Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols
Experimental cells were collected, homogenized, and lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples of 25 μg protein were fractionated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in blocking solution containing rabbit primary antibodies against KEAP1 (D6B12, Cell Signaling Technology, Boston, MA, USA). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase- (HRP-) conjugated anti-goat or rabbit IgG (1 : 2000; Santa Cruz Biotechnology, Inc.) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE ImageQuant.
+ Open protocol
+ Expand
2

Retinal Cofilin Signaling Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Retinas were lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Samples (25 µg) were separated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% non-fat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in the blocking buffer containing rabbit primary antibodies against cofilin, phospho-cofilin, and phospho-LIMK (all from Cell Signing Technology). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Scientific). The images of western blots were captured by GE imageQuant. Relative band intensities were analyzed using Image J software and normalized to non-phospho-blots and to GAPDH.
+ Open protocol
+ Expand
3

Protein Assessment via Cell Extraction

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell extractions for protein assessment studies were performed as previously described [12 (link)]. Briefly, cells were lysed with Cell Lysis Buffer (#9803, Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was standardized by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples (25 μg) were separated by SDS-PAGE in 4% – 20% gradient Tris-glycine precast gels (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% non-fat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4 C in the blocking buffer containing rabbit primary antibodies against CD36 (Abcam, ab133625, 1:500). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE imageQuant imager. Relative band intensities were analyzed using Image J software and normalized to GAPDH.
+ Open protocol
+ Expand
4

Western Blot Analysis of KLF4 in Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols
Adult mice of indicated genotypes were killed, and retinas were dissected and lysed with lysis buffer (Cell Signaling Technology) containing 0.5 mm phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Samples (25 μg) were separated by SDS-PAGE in 4-20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated for 1 h in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in the blocking buffer containing rabbit primary antibodies against KLF4 (1:50; Ab72543, Abcam). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of Western blots were captured by GE ImageQuant. Relative band intensities were analyzed using ImageJ software and normalized to GAPDH.
+ Open protocol
+ Expand
5

KEAP1 Expression in Post-cardiac Arrest Myocardium

Check if the same lab product or an alternative is used in the 5 most similar protocols
Experimental rats were sacrificed 12 hours after ROSC. Then, the rat hearts were harvested and the left ventricular myocardial tissue was collected, homogenized, and lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples of 25 μg protein were fractionated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by an overnight incubation at 4°C in blocking solution containing rabbit primary antibodies against KEAP1 (D6B12, Cell Signaling Technology, Boston, MA, USA). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase- (HRP-) conjugated anti-goat or rabbit IgG (1 : 2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of Western blots were captured by GE imageQuant.
+ Open protocol
+ Expand
6

Protein Assessment via Cell Extraction

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell extractions for protein assessment studies were performed as previously described [12 (link)]. Briefly, cells were lysed with Cell Lysis Buffer (#9803, Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was standardized by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples (25 μg) were separated by SDS-PAGE in 4% – 20% gradient Tris-glycine precast gels (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% non-fat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4 C in the blocking buffer containing rabbit primary antibodies against CD36 (Abcam, ab133625, 1:500). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE imageQuant imager. Relative band intensities were analyzed using Image J software and normalized to GAPDH.
+ Open protocol
+ Expand
7

Quantitative Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
SDS-PAGE and immunoblotting were performed according to the method as described previously [7 (link),25 (link),26 (link)]. Total proteins (20–30 μg) were diluted in Laemmli buffer containing 2-mercaptoethanol, heated to 95°C for 5 min, separated on a 4-20% gradient Tris-glycine precast gel (Invitrogen) at 120 V for 1.5 h. Blots were incubated with primary antibodies specific for NP1 (1:500, BD Transduction Laboratories, Tamecula, CA, USA). HRP (horseradish peroxidase)-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA) were used at 1:10000 dilutions for 1 h at room temperature. The HRP reaction product was visualized using an ECL Western blotting detection kit (GE healthcare). Image films were scanned in gray scale (HP Scanjet G4010) at a high resolution as TIFF files. Immunoreactive protein bands corresponding to the correct molecular mass of target protein were quantified by drawing rectangle around the individual band and the intensity was measured by densitometry using NIH ImageJ software. Values were normalized to internal standard actin, which also serve as a loading control, to make relative comparisons.
+ Open protocol
+ Expand
8

Western Blot Analysis of Mitochondrial Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
SDS/PAGE and immunoblotting were performed according to the method as described previously [10 (link),26 (link)]. Total proteins (20–30 μg) were diluted in Laemmli buffer containing 2-mercaptoethanol, heated to 95°C for 5 min and separated on a 4-20% gradient Tris-glycine precast gel (Invitrogen) at 120 V for 1.5 h. Blots were incubated with primary antibodies for PGC 1α (1:1000; Abcam), TFAM (1:1000; Sigma, St Louis, MO, USA), HSP60 (1:1000; Cell Signaling, Beverly, MA, USA), COX IV (1:1000; Cell Signaling) and actin (1:5000, mouse monoclonal anti b-actin antibody; Sigma.). HRP (horseradish peroxidase)-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, U.S.A.) were used at 1:10000 dilutions for 1 h at room temperature. The HRP reaction product was visualized using an ECL Western blotting detection kit (GE healthcare). Image films were scanned in gray scale (HP Scanjet G4010) at a high resolution as TIFF files. Immunoreactive bands corresponding to the correct molecular mass of target protein were quantified by drawing rectangle around the individual band and the intensity was measured by densitometry using NIH ImageJ software. Values were normalized to internal standard actin, which also serve as a loading control, to make relative comparisons.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!