Protein was isolated from approximately one million cells using RIPA buffer and quantitated using the Pierce BCA Protein Assay kit protocol (Thermo Fisher Scientific). SDS-PAGE was performed using 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (BIO-RAD), and resultant proteins were transferred to nitrocellulose membrane (Thermo Fisher Scientific). Rabbit α-Histone H3 and Rabbit α-cleaved caspase 3 were obtained from Cell Signaling Technology, rabbit α-Phospo-c-Jun was obtained from Abgent, and goat α-rabbit-HRP conjugate secondary antibody was obtained from Abcam. Blots were blocked in appropriate blocking solution according to antibody product insert (5% milk in PBST for α-H3 and α-cleaved caspase 3; 3% BSA in PBST for α-Phospho-c-Jun). Following secondary antibody incubation, blots were developed using SuperSignal West Pico Chemiluminescent Substrate Solution and CL-X Exposure Film (both Thermo Fisher Scientific).
Supersignal west pico chemiluminescent substrate solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
SuperSignal West Pico Chemiluminescent Substrate solution is a laboratory reagent used for the detection of proteins in Western blot analysis. It is a liquid substrate that emits light when it reacts with proteins labeled with horseradish peroxidase (HRP).
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs system, by Bio-Rad (3 mentions)
Perfectblue electro blotter, by Avantor (2 mentions)
Image studio lite version 5, by LI COR (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Pperk1 2, by Cell Signaling Technology (1 mentions)
α tubulin, by GE Healthcare (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
P s6k, by Cell Signaling Technology (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P 4ebp1, by Cell Signaling Technology (1 mentions)
10 protocols using supersignal west pico chemiluminescent substrate solution
1
Protein Expression and Apoptosis Analysis
2
Western Blot Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blot analysis, the mProx24 cells were washed twice with cold PBS, and cell lysate was prepared using RIPA buffer (Wako, Osaka, Japan). The total proteins (40 μg) were separated on 10% SDS/PAGE, then transferred onto a polyvinylidene difluoride membrane (Bio‐Rad, Hercules, CA, USA). The membranes were blocked with 3% skim milk for 1 h at room temperature, then incubated with primary antibodies overnight at 4 °C. The proteins were extracted and blotted with antibodies to α‐tubulin (1 : 4000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), HO‐1 (1 : 2000; Abcam, Cambridge, UK), Nrf2 (1 : 2000; Santa Cruz Biotechnology), p38, phosphoinositol‐p38 (p‐P38) (1 : 1000; Cell Signaling Technology, Beverly, MA, USA), Bcl‐2 (1 : 2000), caspase‐3 (1 : 2000), phosphoinositol‐Erk (p‐Erk)‐1/2, and p‐p‐Erk‐1/2 (1 : 1000; Cell Signaling Technology). Appropriate HRP‐conjugated secondary antibodies were applied for 1 h at room temperature. The proteins were detected with SuperSignal West Pico Chemiluminescent Substrate solution (Thermo Scientific, Rockford, IL, USA). Image Quant LAS 4000 (GE Healthcare, Little Chalfont, Buckinghamshire, UK) was used to measure the relative optical density of each specific band obtained, and α‐tubulin was used to normalize protein loading.
3
Western Blot Analysis of Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were resuspended in loading buffer (300 mM TRIS, 300 mM dithiothreitol, 60% V/W glycerol, 0,3% V/W bromphenol blue, pH 6.8) were denatured by boiling for 5 min before they were loaded on 8% acrylamide gels for separation by SDS-PAGE method [39 (link)]. Separated proteins were electroblotted on the nitrocellulose membrane (Bio-Rad Laboratories), which were subsequently incubated in the BS at room temperature for one hour. The primary antibodies, rabbit anti-PC, were diluted in BS to a final ratio of 1:500 at 4 °C for overnight. After three washing steps, the solution of mouse anti-rabbit conjugated with POD in BS (final dilution ratio 1:10,000) was applied. After one hour of incubation at room temperature, the membrane was washed three times, and subsequently, a chemiluminescent signal was developed after soaking in SuperSignal West Pico Chemiluminescent Substrate solution (Thermo-Scientific) and recorded by Chemidoc XRS system (Bio-Rad Laboratories). Two loading controls were performed by visualization of β-actin (Santa Cruz Biotechnology) and GAPDH (Santa Cruz Biotechnology).
4
Western Blot Analysis of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The recombinant proteins were separated by denaturing SDS-PAGE in 12.5% gels, then transferred onto nitrocellulose membranes by semi-dry blotting at 0.8 mA/cm2 for 1 h (PerfectBlueTM Electro Blotter, Peqlab, Erlangen, Germany). Membranes were blocked with 5% skim milk powder in Tris-buffered saline (TBS) supplemented with 0.05% (v/v) Tween 20 (TBST) for 1 h. Specific mAbs or sera were appropriately diluted in TBST and incubated on the membranes for one hour at room temperature. The membranes were washed three times with TBST and incubated for another hour with anti-murine IgG or gallid IgY HRPO-labeled secondary antibody conjugate (Santa Cruz Biotechnology, Heidelberg, Germany). Blots were washed three times and incubated with SuperSignal™ West Pico Chemiluminescent substrate solution (ThermoScientific, Braunschweig, Germany) before being analyzed on an imaging system (VersaDoc, Bio-Rad, Munich, Germany). Photos were edited with respect to contrast and brightness and composite blots were assembled using cut-out lanes (GIMP software, version 2.10.32).
5
E2 Protein Secretion and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum-free culture media containing the secreted forms of the E2 protein or E2 mutant were collected 3 days after the transfection of Huh7 cells. The samples were treated with SDS sample loading buffer, heated at 70°C for 5 min, and then loaded into the wells of a 4–12% NuPAGE Bis-Tris precast polyacrylamide gel (Life Technologies). After separation by gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane according to the manufacturer’s instructions. Five percent nonfat dry milk in PBS-T was used to block the membrane for 1 h at room temperature. The blocked membrane was then incubated with 1H8 (1∶1000 dilution), as a primary antibody, for an additional hour. After washing, a horseradish peroxidase-conjugated anti-mouse IgG antibody (1:3000 dilution; KPL, Gaithersburg, MD) was used as the secondary antibody. SuperSignal West Pico chemiluminescent substrate solution (Thermo Scientific) was applied to the membrane according to the manufacturer’s protocol. The data were collected with a FluorChem E Imager (ProteinSimple, Santa Clara, CA).
6
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were separated by SDS-PAGE in 12.5% gels. For denaturing conditions samples were heated in Laemmli loading buffer containing dithioerythritol (DTE). Proteins were also separated under non-denaturing conditions in 10% PAGE without SDS and DTE. Proteins were transferred onto nitrocellulose membranes by semi-dry blotting at 0.8 mA/cm2 for 1 h (PerfectBlueTM Electro Blotter, Peqlab, Erlangen, Germany). Membranes were blocked with 5% skim milk powder in Tris-buffered saline (TBS) supplemented with 0.05% (v/v) Tween (TBST) for 1 h. Specific antibodies and sera were appropriately diluted in TBST and incubated on the membranes for 1 h at room temperature. Membranes were washed three times with TBST and incubated for another hour with POD-labeled secondary antibody. Blots were washed again for three times, then incubated with substrate SuperSignal™ West Pico Chemiluminescent substrate solution (ThermoScientific, Braunschweig, Germany) before being analyzed with an imaging system (VersaDoc, Bio-Rad). Photos were edited with respect to contrast and brightness and composite blots were assembled using cut-out lanes (GIMP software).
7
Regulation of mTOR Signaling by Baicalin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sorted naive CD4+T cells were cultured for 3 h with 5 ng/ml TGF-β, 30 U/ml IL-2, and 50 ng/ml anti-CD3/anti-CD28 antibody with or without 40 μM Baicalin or 10 μM MHY1485. Cells were lysed, and proteins were extracted and blotted with antibodies to p-mTOR (Ser2448), mTOR, p-4EBP1, 4EBP1, p-S6K, S6K, and GAPDH (all from Cell Signaling Technology, Beverly, MA). The protein expressions of p-mTOR (Ser2448), mTOR in spleens of MRL/lpr with or without Baicalin were also analyzed. The proteins were detected with SuperSignal West Pico Chemiluminescent Substrate solution (Thermo Scientific, Rockford, IL).
8
C3P3 Enzyme Immunoblotting in HEK-293 and CHO-K1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For C3P3 immunoblotting, HEK-293 and CHO-K1 cells were transfected with the pCMV-FLAGx3-NP868R-(G4S)2-K1ERNAP(R551S) plasmid, which encodes for the FLAGx3 tag fused in frame to the amino-terminal ends of the NP868R-(G4S)2-K1ERNAP(R551S) C3P3 enzyme. Cells were lysed in 200 μl of CLR buffer and lysate was clarified by spinning for 15 s at 12 000 × g at room temperature. Twenty milligrams of total protein were resolved on 4–12% NuPAGE SDS-polyacrylamide gradient gel (Life Technologies, Carlsbad, CA, USA), and subjected to western blotting onto nitrocellulose Hybond membrane (GE Healthcare, Pittsburgh, PA, USA) overnight at +4°C.
Membranes with transferred proteins were blocked with 5% skim milk powder in PBS, then incubated with the rabbit polyclonal F7425 anti-FLAG primary antibody (1:1000; Sigma-Aldrich, Saint-Louis, MO, USA), then with anti-rabbit IgG-conjugated horseradish peroxidase NA9340V antibody (1:10000; GE Healthcare). Bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate solution (Thermo Scientific) and scanned with the Fusion XPRESS gel imager (Vilber Lourmat, Marne-la-Vallée, France). Molecular weights were determined using the Novex Sharp Pre-stained Protein Standard color markers (Thermo Fisher).
Membranes with transferred proteins were blocked with 5% skim milk powder in PBS, then incubated with the rabbit polyclonal F7425 anti-FLAG primary antibody (1:1000; Sigma-Aldrich, Saint-Louis, MO, USA), then with anti-rabbit IgG-conjugated horseradish peroxidase NA9340V antibody (1:10000; GE Healthcare). Bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate solution (Thermo Scientific) and scanned with the Fusion XPRESS gel imager (Vilber Lourmat, Marne-la-Vallée, France). Molecular weights were determined using the Novex Sharp Pre-stained Protein Standard color markers (Thermo Fisher).
9
Detecting MCC Expression in Brain Cancer Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extracted proteins from brain cancer samples were used to detect MCC expression by the dot blot method. The nitrocellulose membrane was spotted with 2 µg of tumor lysate protein. The membrane was incubated in BS1 at RT for 30 min to block the free binding sites on the membrane. Subsequently, the membrane was incubated with the mixture of rabbit antiserum against MCC diluted 1:250 in BS1 at 4 °C overnight. Afterwards, the membrane was washed three times with TBS-T. Subsequently, the membrane was probed with the solution of anti-rabbit IgG molecules conjugated with horseradish peroxidase (1:10,000) in BS1 for 2 h. The membrane was rinsed 3× in TBS-T for 10 min and 1× in TBS, and subsequently, the membrane was soaked in SuperSignal West Pico Chemiluminescent Substrate solution (Thermo Fisher Scientific). The chemiluminescent signal was recorded by the Chemidoc XRS system (BioRad Laboratories, Bratislava, Slovakia). The obtained chemiluminescent signals for MCC were quantified with the Image Studio Lite version 5.2 software (LI-COR Biotechnology).
10
Dot-Blot Detection of Pyruvate Carboxylase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lysates derived from the prostatic cancers were used to detect pyruvate carboxylase expression by the dot-blot method.
Two μg of lysate proteins were spotted on nitrocellulose membrane and let to be air-dried at RT in the dark. The free binding sites on the membrane were blocked by incubating the membrane in a blocking solution (BS) consisting of tris-buffered saline solution (TBS; 19.8 mM Tris, 136 mM NaCl, with pH adjusted to 7.6) supplemented with Tween 20 (0.08 %) and bovine serum albumin (2 %) at room temperature for 30 min. After that, the membrane was incubated in 10 ml of the mixture of rabbit antibodies against pyruvate carboxylase diluted 1:200 in BS at 4 °C overnight. Subsequently, the membrane was washed three times with TBS supplemented with Tween 20 (TBS-T) for 10 min, and then the membrane was probed with a solution of anti-rabbit IgG molecules conjugated with horse-radish peroxidase (1:200) in BS for 2 h. After rinsing the membrane 3x in TBS-T for 10 min and 1x in TBS, the membrane was soaked in SuperSignal West Pico Chemiluminescent Substrate solution (Thermo-Scientifi c), and the chemiluminescent signal was recorded by Chemidoc XRS system (Bio-Rad Laboratories, USA).
The obtained chemiluminescent signals for PC was quantifi ed with the Image Studio Lite version 5.2 software (LI-COR Biotechnology, Inc., USA)
Two μg of lysate proteins were spotted on nitrocellulose membrane and let to be air-dried at RT in the dark. The free binding sites on the membrane were blocked by incubating the membrane in a blocking solution (BS) consisting of tris-buffered saline solution (TBS; 19.8 mM Tris, 136 mM NaCl, with pH adjusted to 7.6) supplemented with Tween 20 (0.08 %) and bovine serum albumin (2 %) at room temperature for 30 min. After that, the membrane was incubated in 10 ml of the mixture of rabbit antibodies against pyruvate carboxylase diluted 1:200 in BS at 4 °C overnight. Subsequently, the membrane was washed three times with TBS supplemented with Tween 20 (TBS-T) for 10 min, and then the membrane was probed with a solution of anti-rabbit IgG molecules conjugated with horse-radish peroxidase (1:200) in BS for 2 h. After rinsing the membrane 3x in TBS-T for 10 min and 1x in TBS, the membrane was soaked in SuperSignal West Pico Chemiluminescent Substrate solution (Thermo-Scientifi c), and the chemiluminescent signal was recorded by Chemidoc XRS system (Bio-Rad Laboratories, USA).
The obtained chemiluminescent signals for PC was quantifi ed with the Image Studio Lite version 5.2 software (LI-COR Biotechnology, Inc., USA)
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!