cells were fixed in 4% paraformaldehyde in PBS for 1 hour at room temperature. Fixed cells were washed in 50 mM NH4Clfor 25 min, permeabilized for 3 × 10 minutes in 0.05% saponin (Sigma, St. Louis, MO) in Membrane Blocking Solution (Life Technologies, Grand Island, NY) and blocked for 1 hour in Membrane Blocking Solution. Cells were stained using mouse anti-occludin antibody (product T5168, Sigma, St. Louis, MO) at a concentration of 1:200 overnight at 4°C and fluorophore-conjugated goat anti-mouse secondary antibody at a concentration of 1:500 for 1 hour (Molecular Probes) at room temperature. Cells were washed 5 × 5 min in PBS ± DAPI (product D9542, Sigma, St. Louis, MO), washed in distilled water and mounted using Prolong Gold anti-fade reagent (Life Technologies, Grand Island, NY).
Membrane blocking solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Membrane Blocking Solution is a laboratory product designed to reduce non-specific binding in Western blotting and other immunoassay techniques. It is a ready-to-use solution that is applied to membrane surfaces to block unoccupied binding sites, preventing unwanted interactions between the target analyte and the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ecl western blotting analysis system, by GE Healthcare (4 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (4 mentions)
Mini protean 3 cell system, by Thermo Fisher Scientific (4 mentions)
Hrp conjugated α tubulin, by Cell Signaling Technology (4 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor mix, by GE Healthcare (2 mentions)
Mammalian protein extraction buffer, by GE Healthcare (2 mentions)
Phospho smad2 3, by Cell Signaling Technology (2 mentions)
Biotinylated smad2 3, by Cell Signaling Technology (2 mentions)
Tbx20, by Merck Group (2 mentions)
Cdkn1a, by Cell Signaling Technology (2 mentions)
Rabbit anti lmna, by Santa Cruz Biotechnology (2 mentions)
Mouse anti lmna, by Santa Cruz Biotechnology (2 mentions)
Camk2d, by Abcam (2 mentions)
Pdgfrb, by Cell Signaling Technology (2 mentions)
20 protocols using membrane blocking solution
1
Immunofluorescence Staining of Occludin
2
Western Blot Analysis of GBM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GBM cells washed with cold PBS were lysed with radioimmunoprecipitation assay buffer (ThermoFisher) containing phenylmethyl sulfonylfluoride (Sigma-Aldrich) and protease inhibitor cocktail (Cell Signaling). Then, the supernatant was collected, and the protein could be measured roughly after the lysate was centrifuged. After electrophoresed on a 10% SDS-PAGE gel at 120 V for 60 min, proteins were transferred onto Immobilon PSQ PVDF membranes (ThermoFisher) at 100 V for another 60 min. After blocked in membrane blocking solution (Invitrogen) for 1 h at 25°C, blot was mixed with primary antibodies for 12 h at 4°C, which was tested by the corresponding horseradish peroxidase-conjugated secondary antibody. It was incubated with the Pierce ECL kit (SignaGen, Jinan, China). Antibodies include rabbit anti-TBK1 (1 : 1,000 dilution; CST, Boston, MA, USA), β-actin (Clone 13E5; Cell Signaling, #4970, 1 : 2000), STING (Clone D2P2F; Cell Signaling, #13647, 1 : 500), and cGAS (Clone D3O8O; Cell Signaling, #31659, 1 : 500) goat anti-rabbit IgG antibody (1 : 5,000 dilution). The critical molecules in partway were probed with primary antibodies against caspase-3, Bcl-2, P53, P62, p-P62, p-TBK1, cGAS, STING, and LC3.
3
Analyzing FAP-Mediated FGF21 Cleavage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed to analyze the ability of FAP to cleave FGF21 at both N- and C-termini. To this end, 25 μM FGF21 protein was incubated with 125 nM FAP enzyme (R&D Systems) at 37 °C for the prescribed times (0, 30, 60 or 90 min) in the presence or absence of 10 μM of Talabostat. The reaction was stopped by addition of Laemmli buffer (Bio-Rad) and heated at 95 ˚C for 10 min. Samples were subjected to electrophoresis using Mini-PROTEAN TGX 8–16% gel (Bio-Rad) and electro transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked for 30 min with Membrane blocking solution (Invitrogen) and subsequently probed with respective antibody at room temperature for 4 h. Antibodies used were total, and anti N- and C-terminus specific (Eagle Biosciences). Membranes were incubated overnight with respective HRP conjugated secondary antibody (R&D Systems) at 4 °C and washed with TBST 5 times for 20 min each. The membrane was dipped in Clarity ECL solution (Bio-Rad) and imaged by exposing the blot to an X-ray film.
4
Protein Expression Analysis Protocol
5
Western Blot Analysis of NF-κB Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer (Thermo Scientific) containing 1× Halt protease inhibitor (Thermo Scientific). Proteins were extracted from whole cells extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred onto PVDF membranes for western blot analysis. After transfer, the membrane was blocked in membrane blocking solution (Invitrogen) overnight at 4°C. Blot was incubated with primary antibody overnight at 4°C. The primary antibody was diluted in buffer according to manufacturer's specification. The primary antibody was detected by the appropriate horseradish peroxidase-conjugated secondary antibody and followed by incubation with Pierce ECL Western Blotting Substrate (Thermo Scientific). Osteopontin (ab8448), ABIN1/TNIP1 (ab130720), and A/20TNFAIP3 (ab92324) were purchased from Abcam. RelB (sc-226), p65 (sc-372), c-Rel (sc-71), and p50 (sc-7178) were purchased from Santa Cruz Biotechnology.
6
Protein Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To extract the total protein and detect the protein concentration, Radio Immunoprecipitation Assay buffer (Sigma) and Bradford Protein Assay Kit (Takara) were used in this study. 40 µg proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel (Invitrogen) for 90 min. Subsequently, proteins on the gel were moved onto the polyvinylidene fluoride (PVDF) membranes (Millipore) and the membranes were sealed in Membrane Blocking Solution (Invitrogen) overnight at 4°C to reduce nonspecific binding. Then, the membranes were submerged in anti-proliferating cell nuclear antigen (anti-PCNA; Abcam, Cambridge, UK, ab18197, 1:1000), anti-matrix metalloproteinase-3 (anti-MMP-3; Abcam, ab53015, 1:1000), anti-MMP-9 (Abcam, ab38898, 1:1000), anti-ERBB4 (Abcam, ab32375, 1:1000) and anti-GAPDH (Abcam, ab9485, 1:3000) diluted by PBS with 0.05% Tween (PBST) at 25°C. 4 h later, the PVDF membranes were washed by PBST three times and incubated with anti-rabbit IgG-HRP (Abcam, ab205718, 1:5000) for 45 min. Following the washing of the membranes by PBST again, the chromogenic reaction was executed via the ECL HRP Substrate Kit (Millipore) and the ERBB4 expression analysis was conducted via ImageLab software version 4.1 (NIH, Bethesda, MD, USA).
7
Western Blot of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular or exosomal protein samples were obtained using Radioimmunoprecipitation assay (RIPA) Lysis and Extraction Buffer (Thermo Scientific) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Cytoplasmic‐Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) was utilized for subcellular fractionation before lysing if necessary. Lysates were quantified using Bradford reagent (Bio‐Rad), and 30 μg protein sample was loaded onto 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel. The electrophoresis separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were then blocked with Membrane Blocking Solution (Invitrogen) and incubated with specific primary antibodies from Abcam (Cambridge, UK), or Cell signaling technology (Danvers, MA, USA), at 4℃ overnight. Then secondary antibody (Abcam) was applied at 37℃ for 1 h, and the membranes were visualized using Amersham™ enhanced chemiluminescence (ECL) Plex™ system (Amersham Pharmacia, Piscataway, NJ, USA).
8
CLEC-47 Protein Expression and Secretion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The heterologous HA-His- or MBP-HA-His-tagged protein CLEC-47 from C. elegans was expressed in HEK 293 cells, and secretion was confirmed by immunoblotting. Cells were seeded into a 6-well plate 1 day before transfection. pcDNA4-based plasmids were transfected with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). The medium was replaced with Opti-MEM I reduced-serum medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 6 h posttransfection. Cells were collected and lysed with radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific).
Recombinant proteins in total cell lysates and conditioned culture medium were separated on an 8% to 16% Mini-Protean TGX stain-free protein gel (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), which was blocked with a membrane-blocking solution (Invitrogen). HA tag monoclonal antibody (12CA5) (Invitrogen) was utilized for detection. The membrane was developed with an enhanced chemiluminescence (ECL) system, and images were acquired with ChemiDoc imaging systems (Bio-Rad).
The inhibition of secretion was performed by adding manufacturer-recommended amounts of brefeldin A (BFA) or GolgiStop (containing monensin) (BD Biosciences, San Jose, CA, USA) to the changed Opti-MEM I reduced-serum medium.
Recombinant proteins in total cell lysates and conditioned culture medium were separated on an 8% to 16% Mini-Protean TGX stain-free protein gel (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), which was blocked with a membrane-blocking solution (Invitrogen). HA tag monoclonal antibody (12CA5) (Invitrogen) was utilized for detection. The membrane was developed with an enhanced chemiluminescence (ECL) system, and images were acquired with ChemiDoc imaging systems (Bio-Rad).
The inhibition of secretion was performed by adding manufacturer-recommended amounts of brefeldin A (BFA) or GolgiStop (containing monensin) (BD Biosciences, San Jose, CA, USA) to the changed Opti-MEM I reduced-serum medium.
9
Fabrication of a Lateral Flow Test Strip
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The test strip consisted of the following five main elements: a sample pad, detection pad, amplification pad, nitrocellulose membrane, and an absorbent pad. The detection pad and the amplification pad were saturated with a membrane blocking solution (Invitrogen), and then dried at 37°C for 2 h. Then, DNA-AuNPs probe 1 and DNA-AuNPs probe 2 were dispensed onto the detection pad and amplification pad, respectively, using the XYZ-3060 dispensing platform, followed by drying at 42°C for 60 min. The test line and control line on the nitrocellulose membrane were prepared using 2 mg/mL streptavidin and 2 mg/mL control probe solutions, respectively, and then dried for 2 h at 37°C. All components were sequentially laminated onto a polyvinyl chloride backing card with 2 mm overlaps to ensure favorable migration of the test solution across the whole test strip. Finally, 5-mm wide LFTSs were cut from the prepared test strip and stored at 4°C.
10
Western Blot for Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were mixed with NuPAGE LDS Sample Buffer (ThermoFisher NP0007), NuPAGE Sample Reducing Agent (ThermoFisher NP0004) and heated for 10 min at 70 °C. Protein (10ug/lane) was resolved on NuPAGE Novex 4–12% Bis-Tris Protein Gels, 1.0 mm, 15-well (ThermoFisher NP0323BOX) and transferred using a Mini-Trans-Blot Cell onto 0.22Um PVDF membranes (ThermoFisher 88520). Membranes were blocked with Membrane Blocking Solution (ThermoFisher 000105) for 1 hour at room temperature (22 °C). The membranes were then incubated overnight at 4 °C with primary antibodies at the following dilutions: anti-Caspase 3 (CST 9661) 1:1000, anti-PARP2 (CST 9542) 1:1000, anti-Actin (CST 4970) 1:5000. The membranes were then washed four times with PBS and 0.5% Tween20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit NA934) 1:15000 for 1 h at room temperature. The membranes were then washed four times with TBST and were visualized with Lumina Forte Western HRP substrate (Millipore WBLUF0500).
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!