Cells were prepared in a six-well plate for 1 × 106 cells per well and cultured for 3 days. Then cells were centrifuged at 4°C for 10 min at 2000 rpm and the supernatant was collected. After measuring protein concentration using BCA, protein electrophoresis was performed in SDS/PAGE. The PVDF membrane was washed with TBS containing 20% Tween20 (TBST) for 5 min three times, and then blocked at 4°C overnight. After incubation with 4 ml primary antibody diluent (SOX15 antibody, 1:500) at room temperature for 2 h, the membrane was washed with TBST for four times, and then incubated with second antibody for 1 h and washed with TBST for four times. After 1–2 min, ECL developing solution (GE Healthcare, Amersham, United Kingdom) was added on to PVDF membrane. Samples were exposed, photographed, and observed under a microscope.
Ecl developing solution
Manufactured by GE Healthcare
Sourced in United Kingdom
The ECL (Enhanced Chemiluminescence) developing solution is a reagent used in Western blotting and other immunoassay techniques to detect and visualize proteins. It generates a luminescent signal when combined with the targeted protein, allowing for the detection and quantification of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Bioss Antibodies (1 mentions)
Epr3554, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti mouse antibody, by Merck Group (1 mentions)
Modified histone peptide array, by Active Motif (1 mentions)
X ray film, by Fujifilm (1 mentions)
Anti rat hrp, by GE Healthcare (1 mentions)
Rabbit anti insulin, by Santa Cruz Biotechnology (1 mentions)
Rat anti k8, by Developmental Studies Hybridoma Bank (1 mentions)
Rabbit anti glut2 polyclonal, by Merck Group (1 mentions)
1 ml syringe, by BD (1 mentions)
Rat anti hsc70, by Stressgen (1 mentions)
4 protocols using ecl developing solution
1
Quantification of SOX15 Protein Expression
2
Protein Expression Analysis via SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were prepared in a 6-well plate containing 1×106 cells per well, and cultured for 3 days. Then, cells were centrifuged at 4oC for 10min at 2,000 rpm, and the supernatant was collected. After measuring the protein concentration using bicinchoninic acid (BCA), protein electrophoresis was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The polyvinylidene fluoride (PVDF) membrane was washed three times with Tris-buffered saline (TBS) containing 20% Tween 20 (TBST) for 5 min each time and then blocked with dried skimmed milk powder at 4°C overnight.
The primary antibodies used were to S100A8 (Abcam, EPR3554), β-actin (Abcam, ab8227), AKT1 (phosphor 473) (Abcam, EP2109Y), cleaved caspase-3 (Asp175) (Cell Signaling, #9661), protein kinase B (Akt) (pan)(Cell Signaling, #4691), caspase-3 (Bioss, bs-0081R) and GAPDH (Bioss, bsm-0978M). Following incubation with 4 ml of primary antibody in diluent, at room temperature for 2 hours, the membrane was washed four times with TBST, and then incubated with the secondary antibody for 1 hour, and washed four times with TBST. After between 1–2 minutes, enhanced chemiluminescence (ECL) developing solution (GE Healthcare, Amersham, UK) was added to the PVDF membranes. Samples were stained and photographed.
The primary antibodies used were to S100A8 (Abcam, EPR3554), β-actin (Abcam, ab8227), AKT1 (phosphor 473) (Abcam, EP2109Y), cleaved caspase-3 (Asp175) (Cell Signaling, #9661), protein kinase B (Akt) (pan)(Cell Signaling, #4691), caspase-3 (Bioss, bs-0081R) and GAPDH (Bioss, bsm-0978M). Following incubation with 4 ml of primary antibody in diluent, at room temperature for 2 hours, the membrane was washed four times with TBST, and then incubated with the secondary antibody for 1 hour, and washed four times with TBST. After between 1–2 minutes, enhanced chemiluminescence (ECL) developing solution (GE Healthcare, Amersham, UK) was added to the PVDF membranes. Samples were stained and photographed.
3
PARP-1 Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Modified Histone peptide array (Active Motif) was blocked by overnight incubation in the tween 20 containing tris buffered saline (TTBS) buffer (10 mm Tris/HCl pH 8.3, 0.05% Tween-20 and 150 mm NaCl) containing 5% non-fat dried milk at 4 °C. The membrane was then washed once with the TTBS buffer and incubated with 4.0 gm PARP-1 (Trevigen) in PARP binding buffer (10 mm Tris-HCl, pH 8, 140 mm NaCl, 3 mm DTT, and 0.1% Triton X-100) at room temperature for 1 h. The membrane was washed in the TTBS buffer and incubated with anti-PARP antibody (SERUTEC at 1:500 dilution) for 1 hr at room temperature in blocking buffer (5% milk in TTBS). The unbound antibody was washed three times with TTBS, and the membrane was incubated with horseradish peroxidase-conjugated anti-mouse antibody (Sigma, 1:2500) in TTBS for 1 hr at room temperature. Finally, the membrane was submerged in ECL developing solution (GE Healthcare) and the image was captured on X-ray film. Typical exposure times were 0.5–2 min. The images were analyzed using an in-house program (Array Analyze, available at here ).
4
Islet Protein Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated and hand‐picked islets from K8+/+ and K8−/− mice were homogenized with a 1‐mL syringe (BD, Franklin Lakes, NJ, USA) and 30‐G needle (Henke Sass Wolf, Tuttlingen, Germany), and samples were prepared for SDS‐PAGE and Western blotting as described.20 Primary antibodies used were as follows: rabbit anti‐insulin (Santa Cruz Biotechnologies), rat anti‐K8 (Troma I; Developmental Studies Hybridoma Bank), rabbit anti‐K18 (275, kind gift from Professor J.E. Eriksson), rat anti‐Hsc70 (Stressgen Bioreagents, Ann Arbor, MI, USA), rabbit anti‐GLUT2 (Polyclonal; Millipore) and rabbit anti‐MFN2 (Sigma‐Aldrich, St. Louis, MO, USA). Anti‐rabbit HRP (Promega Biosciences, San Luis Obispo, CA, USA) and anti‐rat HRP (GE Healthcare, Little Chalfont, UK) secondary antibodies were used. The signal on PVDF‐membranes was developed with ECL developing solution (GE Healthcare) and further exposed to X‐ray films (Fuji, Tokyo, Japan). The Western blot films were then analysed with IMAGE J software (NIH) for individual band quantification.
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!