Western blot assay was performed using the standard method as briefly described 21 (link),22 (link). Protein samples were collected after cells were lysed and quantified. 20~30 μg/lane protein were separated by 10%~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A wet transfer (Bio-Rad, USA) were then performed to transfer the protein onto polyvinylidene fluoride (PVDF) membranes and blocked with non-fat milk for 1 h. The membranes were incubated with primary antibody MFAP5 (HPA010553,Sigma,Germany), MMP9 (D6O3H, Cell signaling technology, USA), MMP2 (D4M2N Cell signaling technology, USA), vimentin (D21H3, Cell signaling technology, USA), Sanil (C15D3, Cell signaling technology, USA), AKT (11E7, Cell signaling technology, USA), p-AKT (D9E, Cell signaling technology, USA), HIF-1α (D1S7W, Cell signaling technology, USA), α-tubulin (11224, Proteintech, USA), β-actin (20536 Proteintech, USA) at 1:1000 dilution overnight and then with anti-rabbit HRP-linked IgG secondary antibody(7074, Cell signaling technology, USA) at 1:2000 dilution for 1 hour. The immunoreactive bands were visualized with ECL Ultra (New Cell and Molecular Biotech, Suzhou, China). α-tubulin and β-actin was used as an internal control.
Wet transfer
Manufactured by Bio-Rad
Sourced in United States
Wet transfer is a laboratory technique used to transfer proteins or nucleic acids from a gel to a membrane for further analysis. The equipment facilitates the transfer of molecules from the gel to the membrane by applying an electrical current, allowing for the detection and analysis of the transferred molecules.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Cytiva (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Mmp9 d6o3h, by Cell Signaling Technology (1 mentions)
Vimentin d21h3, by Cell Signaling Technology (1 mentions)
Hrp linked anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
α tubulin, by Proteintech (1 mentions)
Immuno blot polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Bioruptor pico, by Diagenode (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti goat igg secondary antibodies, by Agilent Technologies (1 mentions)
β tubulin, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
22 protocols using wet transfer
1
Western Blot Protocol for Protein Analysis
2
Immune Protein Dynamics in Mosquitoes
3
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed in 2% sodium dodecyl sulfate (SDS), 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to the membrane by wet transfer (Bio-Rad Laboratories, Hercules, CA, United States). Each membrane was incubated with TBST (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) and 5% non-fat-blocking milk powder at room temperature for 1 h, then incubated overnight with the primary antibody in a shaking bottle at 4°C. The membrane and HRB-conjugated secondary antibody were incubated at room temperature for 1 h. The membrane was then treated with enhanced chemiluminescence reagent (ECL Kit, Amersham Biosciences, Piscataway, NJ, United States) and the proteins were detected using chemiluminescence technology.
4
Quantification of Protein Expression by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Changes in protein content were determined through Western Blot assays. Cells were collected by trypsinization and cell pellets were mixed and incubated at 4 °C with 200–250 μL of lysis buffer (20 mM Tris-HCl, 5 mM EDTA pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) for 30 min and then centrifuged at 10,000 × g for 20 min. Protein concentration was determined by BCA (bicinchonic acid) assay, and proteins were subsequently separated by polyacrylamide gel electrophoresis for 45 min at 150 V. After separation, the proteins were transferred to nitrocellulose membranes using wet transfer (BioRad, Feldkirchen, Germany; 12 mM Tris-Base, 96 mM Glycine, 20% methanol) for 2 h at 50 V. The membranes were rinsed twice with distilled water and treated with Pierce Western Blot Signal Enhancer according to the manufacturer’s instructions. Membranes were incubated with the primary antibody of interest at the dilutions indicated by the manufacturer. The corresponding secondary antibodies (anti-mouse or anti-rabbit, Amersham, GE Healthcare, Freiburg, Germany) were used at 1:7000 dilution. Densitometry analysis was performed by using the Image J software [53 (link)] in FIJI [54 (link)].
5
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed in 2% sodium dodecyl sulfate (SDS), 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to the membrane by wet transfer (Bio-Rad Laboratories, Hercules, CA, USA). Each membrane was incubated with TBST (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) and 5% non-fat blocking milk powder at room temperature for 1 h, and then incubated overnight with the primary antibody in a shaking bottle at 4°C. The membrane and HRB-conjugated secondary antibody were incubated at room temperature for 1 h. The membrane was then treated with enhanced chemiluminescence reagent (ECL Kit, Amersham Biosciences, Piscataway, NJ, USA), and the proteins were detected using chemiluminescence technology.
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded in six-well plates and transfected the day after (see above). The cells were harvested by using a cell scraper and lysed with Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 5% β-mercaptoethanol, and 0.02% Bromophenol Blue). Obtained samples were sonicated (Bioruptor® Pico, Diagenode) and boiled 8 min at 99 °C. Whole-cell lysate was resolved by SDS-PAGE gels (8 or 15% acrylamide) and transferred onto nitrocellulose membranes (0.2 or 0.45 µm-pores) using wet transfer (Bio-Rad). The membranes were blocked with 5% milk in PBS-Tween 20 0.1% for 1 h and incubated overnight at 4 °C with the corresponding primary antibody. Furthermore, followed by incubation with the appropriate IRDye® Secondary Antibody (LI-COR, 1:10,000) for 1 h at room temperature 20–25 °C. Immunoblot with anti-β-actin or G3PDH antibody was used for standardization of protein loading. Details about antibodies used in this study can be found in Table 2 . Bands were visualized using an Odyssey CLx infrared imaging system (LI-COR Bioscience). All targeted proteins of interest were normalized to the selected housekeeping gene, intensity of the bands were verified within the same linear range and quantification was performed using the ImageJ software.
7
Western Blot Analysis of Brain Tissue after MCAO
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rats were killed on day 21 after MCAO. Brains were lysed in lysis buffer, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%). All samples were transferred to a polyvinylidene fluoride membrane by wet transfer (Bio-Rad Laboratories AG, Fribourg, Switzerland). Membranes were incubated with primary antibodies—anti-PSD95, anti-glutamate receptor 2 & 3, anti-synapsin 1, anti-Bcl-2, anti-Bax, anti-PARP, anti-β-tubulin or anti-GAPDH (Table 1 Table 1
8
Western Blot Analysis of Immune Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer in the presence of protease inhibitors (Roche Diagnostics) and phosphatase inhibitors (Pierce). Protein lysates were separated by electrophoresis in 8% SDS polyacrylamide gels and transferred to nitrocellulose membrane (Amersham Biosciences) via wet transfer (Bio-rad). The membranes were blocked overnight at 4 °C with 10% non-fat milk in PBST before probing with specific primary antibodies, followed by horse-radish peroxidase (HRP)-conjugated anti-mouse, anti-rabbit or anti-goat IgG secondary antibodies (DAKO), respectively. The following commercial primary antibodies were used: β-tubulin (Sigma), IRF3 (Santa Cruz Biotechnology), pIRF3(398) (Cell Signaling) and β-actin (Santa Cruz Biotechnology). Polyclonal antibodies against IBV N were raised in rabbits by this laboratory (Li et al., 2005 (link)).
9
Western Blot Analysis of SECISBP2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS–PAGE was performed on lysates and transferred to nitrocellulose via wet transfer (Bio-Rad). SECISBP2 Polyclonal antibody (12798-1-AP; ProteinTech) was used at a 1:1,000 dilution, and Beta Tubulin Monoclonal antibody (66240-1-Ig; ProteinTech) at 1:20,000 dilution. Horseradish peroxidase–conjugated anti-rabbit IgG secondary (65-6120; Invitrogen) was used at a dilution of 1:40,000. Blots were developed using the SuperSignal West Femto kit (Pierce) or fluorescence imaging according to the manufacturer’s protocol with Amersham Imager 600 (GE Healthcare).
10
Western Blot Analysis of HBeAg
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protocol was used as described previously38 (link), with some modifications. 15 µg of protein extracts were separated on 12% or 15% SDS–polyacrylamide gels and blotted onto nitrocellulose using a wet transfer (Bio-Rad Laboratories, Hercules, California, USA), following the manufacturer's instructions. Blocking was done 1 h at room temperature or overnight at 4 °C with tris-buffered saline (TBS), 0,1% Tween-5% milk. HBeAg and precursors were detected with DAKO anti-core antibody (Agilent Technologies, Santa Clara, California, USA) 1/10 000 for 2 h at room temperature. The membrane was washed 3 times with TBS-Tween and incubated 1 h at room temperature with anti-rabbit secondary antibody linked to horse radish peroxidase (HRP) (1/10 000, Bio-Rad Laboratories, Hercules, California, USA). The membrane was washed 3 times with TBS-Tween and incubated 1 min with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, Massachusetts, 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!