Specimens were collected at different time points after mice have been killed and immediately flash frozen in liquid nitrogen. The specimens were then lysed in ice-cold radioimmunoprecipitation buffer supplemented with phosphatase inhibitor cocktails (Complete Mini and PhosphoStop, Roche), centrifuged and the supernatant was removed for protein quantification (Pierce BCA Protein Assay Kit, Thermo Scientific). Twenty-five to 50 μg of protein was loaded into NuPAGE 4–12% bis-tris gels (Life Technologies) and resolved via electrophoresis, then transferred to Immobilon transfer membranes (Millipore). Membranes were blocked in 5% BSA in TBS-T for 1 h before overnight incubation in primary antibody at 4 °C, and incubated in either mouse or rabbit horseradish peroxidase-conjugated secondary antibodies (1:50,000, Amersham Biosciences) in 2% BSA in TBS-T for 1 h. Membranes were imaged using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and images were captured using a GBOX camera system. Antibodies used are listed in Supplementary Table 4 . Most significant uncropped scans are show in Supplementary Fig. 5 .
Immobilon transfer membrane
Manufactured by Merck Group
Sourced in United States
Immobilon transfer membrane is a laboratory equipment product designed for protein transfer and immobilization. It serves as a support for the transfer of proteins from electrophoresis gels to a membrane, enabling further analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Complete mini, by Roche (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosphostop, by Roche (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Las 4000, by Cytiva (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ready gel, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
58 protocols using immobilon transfer membrane
1
Western Blot Analysis of Protein Expression
2
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Muscle and fat lysates, SDS‐PAGE, and Western blot analyses were prepared and performed as previously described (Kristensen et al., 2007 , 2014 ). In short, protein content and phosphorylation of muscle or fat lysate proteins were measured on samples boiled in Laemmli buffer before being subjected to SDS‐PAGE on self‐cast Tris‐HCl polyacrylamide gels. Proteins were transferred to a PVDF membrane (Immobilon Transfer Membranes; Millipore) by semidry blotting. Membranes were blocked in a washing buffer (10 mM Tris‐base, 150 mM NaCl and 0.25% Tween 20) containing low fat milk protein (2%) or BSA (3%) and then probed with primary antibodies and appropriate secondary antibodies. Some membranes were stripped (buffer containing 100 mM 2‐mercaptoethanol, 2% SDS, and 62.5 mM Tris‐HCl). After checking for successful removal of the primary antibody, the membranes were re‐probed with a new primary antibody.
3
Western Blot Analysis of Apoptosis Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against cytochrome c, poly (ADP-ribose) polymerase (PARP), and β-actin were purchased from Santa Cruz Biotechnology. Antibodies against caspase-8, caspase-9, Fas, TNFR1, TNF-α, DR4, DR5, and p53 were purchased from Abcam (Hong Kong). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Pierce Biotechnology. Equal amounts of cell lysates were separated by SDS-PAGE and electrotransferred onto immobilon transfer membranes (Millipore, Bedford, MA). The membranes were blocked with 5% skim milk and probed with the indicated antibodies. The blots were washed and incubated with an HRP-coupled anti-mouse or anti-rabbit IgG antibody, followed by detection with ECL-enhanced chemiluminescence detection reagents (Amersham Biosciences, Piscataway, NJ). β-actin, α-tubulin or COXII were used as loading controls.
4
Western Blot Quantification of Apoptotic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated using standard techniques and quantitated. Briefly, equivalent amounts of buffered protein samples were electrophoresed in SDS‐PAGE gels and electroblotted (Invitrogen X Cell Gel Box and X cell Blotting Module) onto PVDF membranes (Millipore Immobilon Transfer Membranes). The transfer membrane was blocked (5% milk in TTBS), incubated with primary antibody (anti‐Bcl22 , anti‐Bax3 ), washed (TTBS), incubated with secondary IgG:HRP antibody,7 and washed (TTBS). Antibodies were diluted in 1% bovine serum albumin in TTBS. For protein blot visualization, the membrane was incubated with chemiluminescent substrate (Millipore Immobilon Western Chemiluminescent HRP Substrate). Signal strength densitometry was performed for Bcl‐2 and Bax protein quantification using standard techniques with normalization to the β‐actin signal strength.
5
Silencing mitochondrial genes in 143B cells
6
Immunodetection of TgPiT in Parasite Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunodetection of TgPiT, parasites were lysed by suspension in SDS gel-loading buffer (50 mM Tris-HCl (pH 6.8), 50 mM 2-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) followed by boiling in a water bath. The samples were subjected to SDS-PAGE, and the proteins were then electrophoretically transferred to a membrane (Immobilon Transfer Membranes, Millipore, Bedford, MA). The membrane was immersed in blocking buffer (PBS containing 3% skim milk) for 60 min, and then incubated with rat anti-TgPiT (1:1000) or mouse anti-PiT antibodies (1:500) in the blocking buffer for 60 min. Unbound antibody was removed by washing the membrane six times with blocking buffer. Next, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Amersham Pharmacia Biotech; dilution, 1:10,000) in blocking buffer for an additional hour, before detection by chemiluminescence using ECL-Plus.
7
Protein Extraction and Western Blot Analysis
8
Western Blot Analysis of Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon® transfer membranes with a pore size of 0.45 μm (Millipore). After blocking with 2.5% bovine serum albumin (Sigma) for 1 h, membranes were incubated with specific primary antibodies at 4°C overnight. Membranes were then washed with TBST20 solution and incubated with 2nd antibody conjugated to horseradish peroxidase (HRP) for 1 h. After washing, the protein bands were detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific) and the Fusion Solo system (Vilber, Lourmat, France). Densitometric measurements of band intensity were carried out using Image J free software (NIH, Bethesda, MA, USA).
9
Quantifying Virus-Like Particle Budding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Budding of virus-like particles (VLPs) into cell supernatants was detected by Western blot analyses. Wild-type and mutant VP40 bearing a Strep-Tag were cloned into pTriEx-5 (Novagen) and transfected into cells using TrasnIT-LT1 transfection reagent (Mirus). VLPs were harvested 24 hr post-transfection. Cell culture medium was spun down at 3500 rpm for 20 min to pellet any cells out of the media. The cleared supernatants were then ultracentrifuged at 30,000 rpm with an SW-60 rotor (Beckman) for 2 hr through a 20% (w/v) sucrose cushion-50 mM Tris pH 7.4, 100 mM NaCl. Pelleted VLPs were resuspended in 1X NuPAGE LDS sample buffer (ThermoFisher). Cell lysates were collected by washing cells twice with PBS followed by lysis in CytoBuster. VLPs and cell lysates were then run on SDS denaturing gels, transferred onto polyvinylidene difluoride (PVDF) Immobilon transfer membranes (Millipore), and probed with an anti-Strep-Tag antibody (GeneTex). The relative intensities of the bands were quantified by densitometry with a ChemiDoc MP imaging system (Bio-Rad) and ImageJ. The budding index was defined as the amount of Strep-VP40 in the VLPs divided by the amount in the cell lysate and presented as % of wild-type Strep-VP40.
10
Immunoblot Analysis of Phosphorylated Akt and p38
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 120 min stimulation of B cells isolated from healthy donors by anti-IgG/IgM antibody with or without pre-IFN treatment, cells were lysed with 1x SDS sample buffer (Cell Signaling Technologies) and sonicated. The cell lysates were electrophoresed on 12% polyacrylamide gel and then transferred onto Immobilon transfer membranes (Millipore). The membranes were treated with rabbit anti-phosphorylated Akt (Cell Signaling Technologies), rabbit anti-phoshorylated p38 (Cell Signaling Technologies), and rabbit anti-tubulin antibody (abcam, Cambridge, UK). IRDye 800CW-conjugated goat anti-rabbit (LI-COR Biosciences, Lincoln, NE) was used as a secondary antibody. Fluorescence intensity was measured, and densitometric analysis was performed with an Odyssey Infrared Imaging System (LI-COR Biosciences).
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!