The antibodies were purchased from various companies (see Reagents and Resource: Antibodies). The cell was lysed by Passive Lysis Buffer (25 mm Tris‐HCl, 150 mm NaCl, 1% NP40) containing a protease inhibitor cocktail (Roche). Total protein amount was determined by BCA assay (Thermo) and an appropriate amount of denatured protein with Lammeli loading dye was loaded onto 6% or 10% poly‐acrylamide gel for SDS‐PAGE. After that, PVDF membrane was used for transfer. And TBS‐T buffer containing 5% BSA with 0.02% sodium azide was used in membrane blocking and antibody incubations. For Western Blot, all primary antibodies were used in 1:1000 dilution and all secondary antibodies were used in 1:5000 dilution. The chemiluminescent substrate kit was purchased from GE and Tanon. Bound antibodies were visualized using a chemiluminescent substrate kit and exposed to Tanon 5200 and GE. Final quantification of gel intensity was done by ImageJ and plotted in Prism 8.0.
Chemiluminescent substrate kit
Manufactured by GE Healthcare
The Chemiluminescent substrate kit is a laboratory reagent used to detect and quantify the presence of specific proteins or enzymes in a sample. The kit contains a chemiluminescent substrate that, when combined with the target analyte, produces a light signal that can be measured and analyzed.
Automatically generated - may contain errors
Lab products found in correlation
Hybond p, by GE Healthcare (3 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gapdh antibody, by Merck Group (2 mentions)
Blocking agent, by GE Healthcare (2 mentions)
Prism 8, by GraphPad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Peroxidase conjugated rabbit anti mouse igg, by Merck Group (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Rabbit anti bdnf, by Abcam (1 mentions)
Fluorchem 8800, by Bio-Techne (1 mentions)
Hybond, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti actin, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti npas4, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti hdac5, by Santa Cruz Biotechnology (1 mentions)
5 protocols using chemiluminescent substrate kit
1
Western Blot Protein Detection
2
SDS-PAGE and Immunoblotting of ExeD Secretin
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SDS-PAGE gels (12%) were routinely used to analyze protein samples. For analysis of ExeD secretin, 3–8% Criterion pre-cast polyacrylamide Tris-acetate gradient gels (Biorad) were used. Gradient gel samples were standardized to 0.01 OD600 per lane. For immunoblotting, the proteins were transferred to PVDF membranes (GE Healthcare Life Sciences). Visualization of ExeD was achieved by incubation with the appropriate rabbit antiserum followed by incubation with peroxidase-conjugated mouse anti-rabbit IgG (Sigma). The signal was developed with a chemiluminescent substrate kit (GE Healthcare Life Sciences).
3
Quantifying Hippocampal Stress Proteins
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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.
4
Western Blot Analysis of Hippocampal Proteins
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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/ ).
5
Hippocampal Protein Analysis by Western Blot
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For western blot analyses, the collected hippocampi were placed in RNAlater (Life Technologies) and homogenized in a sodium dodecyl sulfate (SDS) sample buffer. Protein extracts were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (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 antibodies: mouse monoclonal anti-Npas4 (1:5,000, Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-HDAC5 (phospho S498) (1:5,000, Abcam plc, Cambridge, UK), mouse monoclonal anti-HDAC5 (1:5,000, Santa Cruz Biotechnology), and rabbit polyclonal anti-PKD1/2/3 PKC micro antibody (Gene Tex, Inc. CA). 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 hr at room temperature. 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/), and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contents, which were detected using a rabbit anti-GAPDH antibody (1:20,000; Sigma-Aldrich CO. LLC. Japan), were used to ensure that the same amount of protein was loaded in each lane.
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