The anesthetized rats (n = 3 per group) were sacrificed and their PFCs isolated (H. N. Wang et al., 2009 (link)). PFCs were manually dissociated in ice cold RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was measured by bicinchoninic acid (BCA) assay. Electrophoresis was then performed on 12% sodium dodecyl sulfate polyacrylamide gel electrophopresis (SDS-PAGE) with 40–50 mg of total protein added to each lane. The resulting gel was then transferred to a polyvinylidene fluoride (PVDF) film. After blocking with 5% non-fat milk for 2 h, the films were incubated with anti-GluA1 (1:1000, ab31232, Abcam), anti-BDNF (1:1000, ab205067, Abcam), anti-PSD-95 (1:1000, ab18258, Abcam), and anti-β-actin (1:2000, HRP-66009, Proteintech) in antibody diluent (A1800, Solarbio, China) at 4 °C for 24 h. After washing with Tris-buffered saline +0.2% Tween 20 (TBST) buffer three times for 15 min, the films were incubated in secondary antibody, which consisted of anti-PSD-95 and anti-GluA1 (peroxidase Affinipure goat anti-rabbit IgG (H + L), each diluted 1:200, ZB-2305, ZSGB-BIO). The film was then washed again three times in TBST. Finally, we activated the blot using the Western Chemiluminescent HRP-substrate (WBKLS0050, Millipore, Billerica, MA, USA). The blot was scanned and then analyzed via densitometry using ImageJ software (NIH, Bethesda, MD)\.
Hrp 66009
Manufactured by Proteintech
Sourced in China
The HRP-66009 is a lab equipment product. It is a horseradish peroxidase (HRP) conjugate. HRP is an enzyme commonly used in various immunoassay techniques as a detection system.
Automatically generated - may contain errors
Lab products found in correlation
Sa00001 1, by Proteintech (2 mentions)
Sa00001 2, by Proteintech (2 mentions)
Zb 2305, by ZSGB-BIO (1 mentions)
Ab18258, by Abcam (1 mentions)
Wbkls0050, by Merck Group (1 mentions)
Ab31232, by Abcam (1 mentions)
Ab205067, by Abcam (1 mentions)
Pierce ecl western blot analysis substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg sa00001 1, by Proteintech (1 mentions)
P0012, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Tissue grinder, by Wuhan Servicebio Technology (1 mentions)
Ab81271, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
A21030, by Abbkine (1 mentions)
Ab7671, by Abcam (1 mentions)
Tanon 5200, by Thermo Fisher Scientific (1 mentions)
Acc 003, by Abcam (1 mentions)
Bca assay kit, by Roche (1 mentions)
5 protocols using hrp 66009
1
Western Blot Analysis of Synaptic Proteins
2
Quantitative Western Blot Analysis
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Proteins were isolated from the examined tissues using radioimmunoprecipitation assay lysis buffer (P0013C; Beyotime) and quantified using a BCA assay (P0012; Beyotime). Equal amounts of proteins (50–100 µg) were electrophoresed on SDS‐PAGE and transferred to PVDF membranes (ISFQ00010; Merck-Millipore), blocked with 5% nonfat milk for at least 1 h at room temperature, and reacted with primary antibodies including Cas9 (7A9-3A3) mouse mAb (14697S, Cell Signaling Technology, 1:1000) and HRP-conjugated Beta Actin (2D4H5) monoclonal antibody (HRP-66009, Proteintech, 1:5000) overnight at 4 °C. The PVDF membrane was then washed in Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, 1:5000). After washing with TBST, the membrane was treated with the Pierce™ ECL western blot analysis substrate (32109; Thermo Fisher Scientific).
3
Brain Protein Expression Analysis by Western Blot
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Protein preparation and western blotting analysis were performed according to a previous method [36 (link)]. Briefly, the bilateral IC brain tissues were collected and stored at -80°C until use. The rat brain tissues were ground at low temperature with a tissue grinder (Servicebio, China), and then fully lysed with RIPA lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors (Beyotime, China). The supernatant was obtained after low-temperature centrifugation. The protein concentration was determined with a BCA kit (AQ, China). Protein (30 μg) was separated by SDS-PAGE gel and electrotransferred to polyvinyl difluoride (PVDF) membranes (Millipor, China). The PVDF membranes were blocked with a quick blocking solution (Yang Guang Bio, China) for one minute, and then incubated with the following antibodies: CGRP (1:1000, cell signaling technology, #14959, USA), GluN2B (1:2000, Proteintech, 21920-1-AP, China), p-GluN2B-S1303 (1:2000, abcam, ab81271, UK), AC1 (1:1000, abcam, ab69597, UK), β-actin (1:3000, Proteintech, HRP-66009, China), and anti-mouse/rabbit secondary antibodies (1:3000, Proteintech, SA00001-1, SA00001-2 China). The protein bands were visualized with the HRP ECL system, and then analyzed with ImageJ 1.53t software for the gray value.
4
Protein Expression Analysis in Cardiac Cells
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Total protein was extracted from cells or left ventricular tissues. The whole operation was carried out on ice. The collected cells or the ground tissues were lysed with RIPA lysis buffer, which added protease inhibitors. Lysates were centrifuged for 15 min at 12,000 rpm under 4℃. The supernatants were used for Western blotting after determining its protein concentration by Bradford assay (Yeasen, China). The protein was separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking, PVDF membranes were incubated with different primary antibodies overnight at 4℃ or 2 h at 25℃.
Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), CaV1.2 α1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus). The secondary antibodies used were HRP-conjugated goat anti-mouse (0.02 µg/mL, SA00001-1, Proteintech) or anti-rabbit IgG (0.02 µg/mL, SA00001-2, Proteintech), or rabbit anti-goat IgG (A21030, Abbkine). We used an imaging system (Tanon 5200, China) to visualize the blot with the enhanced chemiluminescence reagent (Pierce, Thermo Fisher Scientific).
Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), CaV1.2 α1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus). The secondary antibodies used were HRP-conjugated goat anti-mouse (0.02 µg/mL, SA00001-1, Proteintech) or anti-rabbit IgG (0.02 µg/mL, SA00001-2, Proteintech), or rabbit anti-goat IgG (A21030, Abbkine). We used an imaging system (Tanon 5200, China) to visualize the blot with the enhanced chemiluminescence reagent (Pierce, Thermo Fisher Scientific).
5
Mitochondrial Dynamics in Retinal Tissues
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Retinal tissues were lysed in radioimmunoprecipitation assay buffer (ASPEN, China) containing a protease inhibitor cocktail (ROCHE). All samples were subjected to protein content determination by using a BCA assay kit (ROCHE), electrophoresed by using SDS-PAGE (ASPEN, China), and transferred to a PVDF membrane. Subsequently, the membrane was blocked with 5% skimmed milk at room temperature for 1 h, incubated with the primary antibody diluent at 4 °C overnight, then incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blot was developed by using enhanced chemiluminescence and photographed in a dark room. Immune response bands were analyzed by utilizing Image J, and GAPDH and β-actin were used as the loading controls. The primary antibodies were as follows: Bax (1:1000, Abcam: ab182773), Bcl-2 (1:1000, Proteintech: 60178-1-Ig), cleaved caspase-3 (1:1000, CST: 9661), Bcl-xL (1:1000, CST: 2764); OPA1 (1:1000, CST: 80,471); DRP1 (1:1000, CST: 8570); MFN2 (1:1000, CST: 9482); GAPDH (1:1000, CST: 5174); and β-actin (1:1000, Proteintech: HRP-66,009).
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