TCDD was purchased from Cambridge Isotope (Andover, MA). Anti-phospho-AMPKα (#2535), anti-AMPK (#2532), anti-phospho-ACC (#11818), anti-ACC (#3676), anti-VDAC (#4661 T), anti-COX IV (#4850), anti-Ferritin (#sc-376594), anti-LC3 (#2775), anti-OPA1(#80471), and anti-mouse IgG, HRP-linked secondary antibody (#7076 S), anti-rabbit IgG, and HRP-linked secondary antibody (#7074 S) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-GAPDH (#sc-47724), anti-CCS (#sc-374205), anti-Drp1 (#sc-271583), and anti-GFP antibody (#sc-9996) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FLAG antibody (#F1804), bathocuproinedisulfonic acid (BCS), and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Nile Red, Mitotracker, and Phen Green FL were purchased from Invitrogen (Carlsbad, CA). JetPEI transfection reagent was obtained from VWR International (Radnor, PA, USA). Anti-Mitofusin2 (#ab56889), TMRE, and superoxide dismutase (SOD) kit were purchased from Abcam (Cambridge, MA). CH-223191 was obtained from Tocris (Bristol, UK). TransIT-QR Hydrodynamic Delivery Solution was purchased from Mirus (Pittsburgh, PA, USA). EZ-ATP Assay kit was obtained from DoGenBio (Seoul, Korea). All other chemicals were of the highest grade commercially available. CF4 and Ctrl-CF4 were synthesized as previously reported29 (link).
Hrp linked secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
HRP-linked secondary antibody is a laboratory reagent used in various immunological techniques. It consists of a secondary antibody conjugated with the enzyme horseradish peroxidase (HRP). The HRP-linked secondary antibody is used to detect and amplify the signal from a primary antibody, enabling the visualization and quantification of target proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (15 mentions)
Bca protein assay kit, by Beyotime (14 mentions)
Gel imager system, by Bio-Rad (14 mentions)
Gapdh, by Cell Signaling Technology (12 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
Enhanced chemiluminescence kit, by Santa Cruz Biotechnology (9 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (9 mentions)
Enhanced chemiluminescence kit, by Merck Group (6 mentions)
Ripa buffer, by Beyotime (6 mentions)
Bcl 2, by Cell Signaling Technology (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
C myc, by Cell Signaling Technology (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Chemidoc touch imaging system, by Bio-Rad (5 mentions)
Clarity western ecl substrate, by Bio-Rad (5 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (5 mentions)
Anti cyclin d1, by Cell Signaling Technology (4 mentions)
Bca assay, by Thermo Fisher Scientific (4 mentions)
124 protocols using hrp linked secondary antibody
1
Mitochondrial Dynamics and Metabolism Assay
2
Western Blot Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins from tissue and cell samples were washed three times with PBS. Then the samples were lysed in RIPA lysis buffer for 30 min. The protein lysates were separated by 10% SDS-PAGE. The specific proteins were transferred on PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% non-fat milk in TBST for 30 min and then washed twice with TBST. The specific proteins were incubated with the different primary antibodies overnight at 4°C. The used primary antibodies were as follows: JAK1 (Ab-1022), p-JAK1 (SAB4300123), JAK2 (Ab-570), p-JAK2 (SAB4301238), p-STAT3 (SAB4300034), Ki67 (SAB4501880) and STAT3 (SAB4502871) (Sigma-Aldrich), cleaved caspase-3 (9654), matrix metalloproteinases [MMP-2 (40994) and MMP-9 (15561)], E-cadherin (14472), N-cadherin (4068), Snail (3879) and β-actin (8457) (Cell Signaling Technology, Beverly, MA, USA). The membranes were washed twice and then incubated with HRP-linked secondary antibody (7075; Cell Signaling Technology) for 1–2 h. Protein signals were visualized using enhanced chemiluminescence reagents (ECL; GE Healthcare, Waukesha, WI, USA).
3
Western Blot Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For total protein extraction, cells were disrupted in RIPA (Applygen, Beijing, China) lysis buffer containing a Protease and Phosphatase Inhibitor Cocktail (Beyotime, Shanghai, China) at 4 °C for 30 min. The cell lysates were centrifuged at 12,000 × g for 10 min at 4 °C and the supernatants were collected. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to quantify the protein concentrations of each sample. Protein aliquots were separated on 10% SDS-PAGE and proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Billerica, USA). The blots were then blocked in 5% fat-free milk for 1 h at room temperature and incubated with primary antibodies at the recommended dilutions with gentle agitation overnight at 4 °C. The primary antibodies included SPHK1, ATF3, p-NF-κB-p65, NF-κB-p65, p-STAT3, STAT3, p-p38-MAPPK, p38-MAPPK, p-ERK1/2, ERK1/2, p-JNK, JNK, p-JUN and JUN (Cell Signaling Technology, Danvers, USA), PTX3 and GAPDH (Proteintech, Rosemont, USA). After washing, the blots were incubated with the corresponding HRP-linked secondary antibody (Cell Signaling, Danvers, USA). Stained bands were visualized with a Tanon 5200 Automatic Imaging System (Tanon, Shanghai, China) using a hypersensitive ECL solution (Applygen, Beijing, China).
4
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with cold phosphate-buffered saline (PBS), and lysed on ice in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM Na3VO4, 10mM NaPPi, 10mM beta-glycerophosphate and 50 mM NaF) supplemented with the Halt protease inhibitor cocktail (Thermo Scientific). Protein concentration was measured using BCA protein assay kit (Pierce). Equal amounts of protein were solubilized and heated at 75°C in LDS sample buffer (Invitrogen) with sample reducing agent (Invitrogen) for 10 min, and then separated by SDS-PAGE, and transferred to Immobilon-P membrane (Millipore). Following incubation in blocking buffer (TBS with 5% nonfat dry milk and 0.1% Tween 20) for 1 hour at room temperature, the membranes were probed overnight at 4°C. The membranes were washed, and then probed with an HRP–linked secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. Specific proteins were detected using an enhanced chemiluminescence (ECL) Western blotting detection kit (GE Healthcare) according to the manufacturer's instructions. All Western blot analysis was repeated for at least three times.
5
Apoptosis Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After incubation with CAHB for 24 h, Jurkat cells were washed with PBS, harvested, and lysed in RIPA cell lysis buffer. Quantification of total protein was performed using a BCA kit (Thermo Fisher, USA). We separated 50 μg proteins in each group using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred them to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked by 5% skimmed milk for 1 h at room temperature. The primary antibodies (Cell Signaling Technology, USA) were diluted into indicated concentration (Bcl-2 1: 500, Bax 1: 1000, Akt 1: 1000, p-Akt 1: 1000, GAPDH 1: 1000 A) in 5% skimmed milk and incubated with the PVDF membrane at room temperature for 2 h. After washing 3 times with TBST for 10 min, the membrane was incubated with HRP-linked secondary antibody (1: 2000 dilution) (Cell Signaling Technology, USA), at room temperature for 1 h. Then, the membrane was washed again with TBST 3 times and the signal was detected using an ECL Plus Kit (Applied Biosystems). Quantity One was used for grayscale value calculation of Western blot images.
6
KDM5D Knockdown Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KDM5D Knockdown (KD) and Wildtype (WT) ITSCs were harvested by surface scraping followed by cell lysis on ice with lysis buffer (10 mM Tris, 1% SDS, 3 mM EDTA). Protein concentrations of the samples were adjusted to 20 µg total protein amount and mixed with 4× loading buffer followed by heating at 95 °C for 8 min. Samples were subjected to electrophoresis on 10% denaturing SDS polyacrylamide gel and transferred with a semi-dry blotter to a nitrocellulose membrane (Carl Roth GmbH, Karlsruhe, Germany). Blocking of membrane with 5% milk powder (Carl Roth GmbH, Karlsruhe, Germany) in 1× TBS with 0.05% Tween 20 (Sigma-Aldrich, Taufkirchen, Germany) was followed by incubation with the first antibody against JARID1D/KDM5D (1:500; Cell Signaling Technology Inc., Danvers, MA, USA) in 1× TBS with 5% milk powder and 0.05% Tween 20 while shaking at 4 °C overnight. HRP-linked secondary antibody (1:4000; Cell Signaling Technology Inc.) was applied for 1h at RT. Visualization was performed via enhanced chemiluminescence. Beta-actin antibody (1:2000; Cell Signaling Technology Inc.) was applied as control for 1 h at RT followed by exposure to secondary antibody as described above.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from NSCLC tumor tissues and cells using RIPA lysis buffer containing protease inhibitor cocktail (Invitrogen, USA), then quantified by a BCA Protein assay kit (Solarbio, Beijing, China). 10 ug protein was used for SDS-PAGE electrophoresis. Separated protein in SDS_PAGE gel was transferred onto the PVDF membrane (BioRed, USA). After blocking with 5% skimmed milk for 1 h, the membrane was then incubated with primary antibodies: FGF11 (1:1000; Cell Signaling Technologies #3139, MA, USA), HIF-1α (1:1000, Cell Signaling Technologies #3716), β-Actin (13E5) (1:2000, Cell Signaling Technologies #4970) and GAPDH (1:2000; Cell Signaling Technologies #2118) for 2 h or overnight at 4℃. The membrane was washed 3 times with TBST for 5 min each. After wash, the membrane was further incubated with HRP-linked secondary antibody (1:3000; Cell signaling #7074, MA, USA) at room temperature for 1 h. Then the membrane was washed 4 times with 1 × TBST and the protein bands were visualized using an enhanced chemiluminescence kit (Santa Cruz, TX, USA) and photographed on a gel imager system (Bio-Rad).
8
Western Blot Analysis of Neuroinflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ipsilateral hemispheres of the mouse brain samples were collected and homogenized in whole cell lysis buffer (KeyGEN BioTECH, China). The protein supernatants were centrifuged at 12,000 x g for 10 minutes at 4 °C and denatured at 100 °C. The proteins were subjected to SDS-PAGE (KeyGEN BioTECH, China) and transferred to PVDF membranes (Millipore, USA). Then, the membranes were blocked with 5% skim milk and incubated with a rabbit polyclonal anti-interleukin 1β (IL-1β) antibody (Abcam, UK), a rabbit polyclonal anti-TNFα antibody (Abcam, UK), a rabbit polyclonal anti-brain-derived neurotrophic factor (BDNF) antibody (Abcam, UK) and a rabbit anti-GAPDH antibody (Abcam, UK) overnight at 4 °C, followed by incubation with an HRP-linked secondary antibody (Cell Signaling Technology, USA) for 2 h at RT. Finally, the membranes were exposed and analyzed with ImageJ software.
9
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were rinsed twice with cold PBS and lysed by RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (Roche). Protein (40 μg per sample) was separated by SDS-PAGE using a 10% polyacrylamide gel. The proteins were transferred electrophoretically onto a PVDF membrane. Blotted membranes were blocked in 5% skimmed milk diluted in TBST, followed by incubation with appropriate primary antibodies (anti-EGFR, anti-cyclin D1, CDK4, anti-cyclin D2, anti-p21, anti-p57, anti-MMP-7, anti-MMP-9, anti-cleaved caspase 3, and anti-GADPH; obtained from Cell Signaling Technology and all the antibodies were diluted 1:1000.) overnight at 4°C. The membranes were then washed for 5 minutes for three times with TBST, and subsequently incubated for 1 hour with HRP-linked secondary antibody (Cell Signaling Technology) at room temperature. GADPH was used as an internal control. The blots were detected using an enhanced chemiluminescence kit (Millipore) and subjected to autoradiography using X-ray film.
10
Quantitative Western Blot Analysis of Heparanase and Related Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies used were: Heparanase1/HPA1(1:2000) (Abcam, ab128931), Glypican1/GPC1 (1:1000) (Abcam, ab106003), Syndecan-1/SDC-1 (1:2000) (Abcam, ab128936), ICAM1 (1ug /ml) (Abcam, ab124760), GAPDH (1:2500) (Abcam, ab9485)—Loading Control. HRP-linked secondary antibody (#7074) was from Cell Signaling Technology, Inc.
Total protein was extracted using T-PER™ Tissue Protein Extraction Reagent (Pierce #78510), the concentration and quantitation was measured by Pierce™ BCA Protein Assays (Pierce #23227). Then, the 20 μg total protein resolved by 10% SDS-polyacrylamide gel electrophoresis was transferred to polyvinylidene difluoride (PVDF) membrane (Millipore), blocked with 5% non-fat milk and 0.1% Tween-20 for 1 h, and incubated with primary and secondary antibodies overnight at 4 °C and 1 h at room temperature, respectively. Signals were detected by exposure to X-ray films after treatment with the ECL Western Blotting Substrate kit (Pierce).
Total protein was extracted using T-PER™ Tissue Protein Extraction Reagent (Pierce #78510), the concentration and quantitation was measured by Pierce™ BCA Protein Assays (Pierce #23227). Then, the 20 μg total protein resolved by 10% SDS-polyacrylamide gel electrophoresis was transferred to polyvinylidene difluoride (PVDF) membrane (Millipore), blocked with 5% non-fat milk and 0.1% Tween-20 for 1 h, and incubated with primary and secondary antibodies overnight at 4 °C and 1 h at room temperature, respectively. Signals were detected by exposure to X-ray films after treatment with the ECL Western Blotting Substrate kit (Pierce).
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!