For Western blot analysis, collected hippocampi were placed in RNAlater (Life Technologies) and homogenized in an SDS sample buffer. Protein extracts were separated by SDS–polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (Hybond P; GE Healthcare). The membrane was blocked with a blocking agent (GE Healthcare) and then incubated at 4°C overnight with a primary antibody for rabbit anti-HSP110 (1:5000, StressMarq Biosciences Inc.), rabbit anti-HSP25 (1:5000, Cosmo Bio Co. Ltd.), mouse anti-HSP60 (1:1000, Cosmo), mouse anti-HSP72 (1:1000, Cosmo), mouse anti–phospho-CREB (Ser133) (1:5000, Cell Signaling Technology), rabbit anti-CREB (Ser133) (1:5000, Cell Signaling), and rabbit anti-BDNF (1:5000, Abcam). After washing with tris-buffered saline containing 0.1% (v/v) Tween 20, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (1:20,000) for 1 hour at room temperature. The antibody-reactive bands were visualized using the chemiluminescent substrate kit (GE Healthcare). Bands were analyzed by densitometry using FluorChem 8800 (Alpha Innotech), and the content of GAPDH, which was detected using a rabbit anti-GAPDH antibody (1:20,000; Sigma), was used as a control to ensure that the same amount of protein was loaded in each lane.
Blocking agent
Manufactured by GE Healthcare
Sourced in United States, Germany, United Kingdom
A blocking agent is a reagent used in various laboratory techniques, such as Western blotting and enzyme-linked immunosorbent assays (ELISAs), to prevent non-specific binding of antibodies or other proteins to the test surface or membrane. Blocking agents help to reduce background signal and improve the specificity and sensitivity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (3 mentions)
Chemiluminescent substrate kit, by GE Healthcare (2 mentions)
Hybond p, by GE Healthcare (2 mentions)
Rabbit anti gapdh antibody, by Merck Group (1 mentions)
Rabbit anti bdnf, by Abcam (1 mentions)
Fluorchem 8800, by Bio-Techne (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue, by Merck Group (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Anti rabbit secondary antibody, by Merck Group (1 mentions)
Westernbright ecl detection kit, by Advansta (1 mentions)
Anti lc3b antibody, by Nanotools (1 mentions)
Enhanced chemiluminescence, by PerkinElmer (1 mentions)
Fla 2000, by Fujifilm (1 mentions)
Biodoc it system, by Analytik Jena (1 mentions)
Trans blot semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Hybond n, by GE Healthcare (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
10 protocols using blocking agent
1
Quantifying Hippocampal Stress Proteins
2
Far-Western Blotting for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used the Yuliang Wu far‐western blotting method35 ; 20 μg of all purified proteins (RBD, hCypA, and ACE2) was loaded into wells with 62.5 mM Tris–HCl pH 6.8, 10% glycerol, 1% SDS, 1% β‐mercaptoethanol and 0.01% bromophenol blue for 5 min at 95°C. Total proteins were separated using 4%–12% SDS‐PAGE at 120 mA for 2 h and transferred to a Polyvinylidene Fluoride (PVDF) membrane (Amersham, USA) at 100 V for 2 h. The membrane was stained with Coomassie Brilliant Blue and Ponceau S (Sigma, USA) to determine whether the proteins had transferred from the gel to the membrane. Next, the proteins were denatured and refolded on the membrane in the AC buffer (100 mM NaCl, 20 mM Tris–HCl pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1% Tween‐20, 2% skim milk, and 1 mM DTT) by gradually reducing the guanidine–HCl concentration. The membrane was then blocked with 5% (w/v) blocking agent (GE Healthcare, USA) for 1 h at RT and incubated with 10 μg purified “bait” RBD protein RBD in PBS overnight at 4°C. The membranes were washed five times with PBST buffer (PBS containing 0.1% [v/v] Tween 20) and incubated with anti‐RBD antibody for 2 h at 4 °C. After incubation, the membrane was washed with PBST buffer three times and probed with an anti‐rabbit secondary antibody (Sigma, USA) for 1 h at RT. Immunoreactive proteins were detected using a WesternBright ECL detection kit (Advansta, USA).
3
Western Blot Analysis of LC3B in MEFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MEFs were washed three times with PBS and then incubated for 10 min in cold lysis buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100 and a protease-inhibitor cocktail (Roche)). After 10 min of rotation on a wheel, cell lysates were centrifuged for 15 min at 13,000 RPM at 4°C to sediment cell debris. Protein concentration of these clear lysates was determined using the BCA (Bicinchoninic acid) protein assay (Pierce). Fifteen micrograms of proteins were separated by SDS-PAGE 12% and then, transferred onto polyvinyl difluoride (PVDF) membranes. Membranes were blocked for 1 h in PBS containing 0.1% Tween 20 and 2% of blocking agent (GE Healthcare), then incubated for 2 h with a primary monoclonal anti-LC3B antibody (NanoTools, Germany) and a secondary anti-mouse antibody conjugated to horseradish peroxidase (HRP). The activity of HRP was revealed by enhanced chemiluminescence (Perkin-Elmer).
4
Native PAGE Separation and Detection of RNA:DNA Hybrids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Size separation of nucleic acids under native conditions was performed using 15% native PAGE gels, pre-run for 1 h at 5 W 4°C, samples were loaded in an equal volume of native loading buffer (30% (v/v) glycerol, 80 mM HEPES-KOH (pH 7.9), 100 mM KCl, 2 mM magnesium acetate) and electrophoresed in 1× TBE at 5 W 4°C in a vertical gel tank (EV200, Cambridge Electrophoresis). Nucleic acids were visualised using Sybr Gold (Invitrogen) according to manufacturer's instructions and imaged on a phosphorimager (FLA-2000, Fujifilm) or UV transilluminator (BioDoc-It System, UVP). Immunoblot detection of RNA:DNA hybrids was performed by transfer of electrophoresed nucleic acids to Hybond-N+ (GE Healthcare) using a Trans-Blot® Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories), in 1× TBE for 30 min at a maximum current of 3 mA/cm2. Nucleic acids were cross-linked to the membrane by exposure to 1200 mJ/cm2 UV (Stratalinker, Stratagene). Membrane was pre-blocked overnight in 1% (w/v) blocking agent (GE Healthcare) in 0.1% (v/v) Tween-20/PBS at 4°C, washed in 0.1% (v/v) Tween-20/PBS, probed with 200 ng/ml S9.6 antibody in 2% (w/v) BSA/PBS overnight at 4°C, and then incubated with HRP-conjugated goat anti-mouse IgG antibody (1:5,000, Dako Corporation) for 1 h at room temperature, followed by ECL detection (GE Healthcare).
5
Western Blot Analysis of Hippocampal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously described9 (link). Briefly, dorsal hippocampal samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (HybondP, GE Healthcare UK Ltd.). The membrane was blocked with a blocking agent (GE Healthcare) and then incubated at 4 °C overnight with the following primary and secondary antibodies: mouse monoclonal anti-MAOB (1:1,000; Santa Cruz Biotechnology, Inc., sc-515354), mouse monoclonal anti- TIEG2 (KLF11) (1:1,000; Santa Cruz Biotechnology, sc-136101), rabbit polyclonal anti-p-HP1γ (Ser83) (1:5,000; Invitrogen, Thermo Scientific, PA517210), mouse monoclonal anti-HP1γ (1:5,000; Santa Cruz Biotechnology, sc-398562), mouse monoclonal anti-actin (1:10,000; Santa Cruz Biotechnology, sc-8432), horseradish peroxidase-conjugated secondary antibody against mouse or rabbit (1:20,000; Santa Cruz Biotechnology, sc-2357 for mouse, sc-2004 for rabbit). The antibody-reactive bands were visualized using a chemiluminescent substrate kit (GE Healthcare). Bands were analyzed by densitometry, using ImageJ (https://imagej.nih.gov/ij/ ).
6
Protein Expression Analysis in Cardiac Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate protein expression levels, left ventricular tissue was homogenized with a Polytron homogenizer in lysis buffer and protease inhibitor cocktail (Roche). Fifty micrograms of total protein was separated by 10% SDS-PAGE. Separated proteins were electrotransferred to nitrocellulose membranes. Membranes were blocked with 5% blocking agent (Amersham, GE healthcare) and immunoblotted using the following antibodies: Col I and Col III, and GAPDH was used as a load control (Santa Cruz Biotechnology). After that, a secondary horseradish peroxidase-conjugated antibody was applied for 1 hour at room temperature. The blots were developed with a chemiluminescence detection system (Amersham, GE Heath care) and visualized by exposure to Kodak radiographic film. Density of bands was measured with Image J (National Institutes of Health, Bethesda, MD, USA).
7
FcRn Quantification by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following SDS-PAGE proteins were transferred onto 0.45μm Nitrocellulose membrane (GE Healthcare) in Tris/glycine buffer (25mM Tris and 190mM glycine) at 150mAmps overnight. Membranes were blocked with 5% blocking agent (GE Healthcare) in PBS/0.1% Tween-20 for 1 hour. After blocking, the membranes were washed three times for 5 min with PBS/0.1% Tween-20. Membranes were incubated with a primary antibody: B8 anti-FcRn mouse monoclonal IgG2a (Santa Cruz Biotechnology, sc-271745) diluted 1:1000 in 1% BSA/PBS/0.1% Tween-20 for 1 hour. Membranes were washed six times for 5 min with PBS/0.1% Tween-20 and then incubated with secondary antibody: goat anti-mouse IgG F(ab’)2-HRP (Jackson ImmunoResearch Laboratories, Stratech, 115-036-072) diluted 1:2000 for 1 hour. Membranes were washed six times for 5 min before being treated with ECL Select (GE Healthcare). Immunoblots were visualised using a charge-coupled device (CCD) camera system; Image Quant (GE Healthcare). Quantification of FcRn was carried out using Image Quant TL software (GE Healthcare). A standard curve was generated from known amounts of purified recombinant human FcRn extracellular domain (ECD) (courtesy of UCB). The difference in molecular weight between the ECD and endogenous FcRn was taken into account in the quantification.
8
Western Blot Analysis of Cardiac Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate protein expression levels, left ventricular tissue was homogenized in lysis buffer with the protease inhibitor cocktail included (Roche). Fifty micrograms of total proteins were separated by 10% SDS-PAGE. Separated proteins were electrotransferred to nitrocellulose membranes. Membranes were blocked with 5% blocking agent (GE healthcare, Amersham, Buckinghamshire, UK) and immunoblotted using the following antibodies: TIMP-1 (Catalog No. 21734 Santa Cruz Biotechnology), TIMP-4 (Catalog No. 30076 Santa Cruz Biotechnology), MMP-2 (Catalog No. 10736 Santa Cruz Biotechnology), MMP-9 (Catalog No. 6841R Santa Cruz Biotechnology), GAPDH was used as a load control (Catalog No. 25778 Santa Cruz Biotechnology). After that, secondary horseradish peroxidase-conjugated antibody was applied for 1 hour at room temperature. The blots were developed with a chemiluminescence detection system (GE Heathcare, Amersham, Buckinghamshire, UK) and visualized by exposure to Kodak radiographic film. Density of bands was measured with Image J (National Institutes of Health, Bethesda, MD, USA).
9
Verifying Immune Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two of the identified immune-related proteins, Cyclophilin A (CypA) and Cofilin 1 (Cof1), that were included in the list of proteins contributing to the separation of the TDI-treated group and AOO-treated group in the PCA were verified via Western blotting in B lymphocytes obtained from a new, independent set of similarly treated TDI- and AOO-treated mice (n = 10/group). Briefly, proteins were loaded and separated on 4–12% Bis/Tris Midi-gels (Invitrogen, Merelbeke, Belgium) and subsequently transferred to a PVDF membrane (iBlot, Gel Transfer Stack, Invitrogen). Membranes were blocked (1–2 h, 5% blocking agent, GE Healthcare) and incubated overnight with primary antibody (LSP-1: 1/1000, goat Ab, Santa Cruz Biotechnology; CypA: 1/1000, mouse Ab, Santa Cruz Biotechnology; GAPDH, internal standard, 1/200000, mouse Ab, Dako). Following secondary Ab incubation (LSP-1: 1/50000, donkey anti-goat IgG, Santa Cruz Biotechnology; Cof1 and GAPDH: 1/100000, goat anti mouse IgG, Dako) protein bands were visualized using chemiluminescence detection (Supersignal West Dura, Thermo Scientific) on ECL hyperfilm (GE Healthcare). The protein bands were semiquantitatively evaluated by densitometry (ImageQuant TL v2009, GE Healthcare).
10
Immunoblotting Analysis of THP-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THP-1 cells (2 × 105 cells/mL) were treated with 200 μg/mL of raw milk-derived EVs, PT-peptide, or EVs-PT peptide for 72 h. After treatment, the THP-1 cells were rinsed with ice-cold PBS and lysed for 20 min on ice using RIPA lysis buffer containing protease and phosphatase inhibitors. The cells were centrifuged at 12,000 ×g for 10 min at 4 °C. Protein extracts (20 μg) were resolved using 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to polyvinyldiene fluoride membranes (80 V, 120 min). The membranes were blocked with a 5% blocking agent (GE Healthcare Bio-Sciences) in Tris buffered saline containing 0.1% Tween-20 (TBST) for 1 h, and incubated with the primary antibody against Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab8245; 1:2000dilution) or CD11b (ab133357; 1:1000 dilution) overnight at 4 °C. Subsequently, the membranes were washed three times with TBST and incubated with appropriate horseradish peroxidase conjugated secondary antibodies, followed by analysis with an enhanced chemiluminescence (ECL) plus Western blotting detection system (GE Healthcare Bio-Science). GAPDH expression was assessed as an internal reference for normalization.
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!