Transfected cells were harvested and protein was extracted using the RIPA Lysis Buffer (Kaiji, Nanjing, China) followed the protocol provided by the manufacture. The quantitative of protein was measured by the BCA Protein Assay Kit (Kaiji, Nanjing, China). Then equal amount of proteins were loaded in the SDS page gel and transferred onto a PVDF membrane after the electrophoresis. After blocked with defatted milk for 2 h, the membrane was incubated all night with antibodies against SNAP23 (Abcam, ab42483 1:1000) or GAPDH (CST,2118, 1:1000). After washing with TBST 3 times, the membrane was incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:10,000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1:10,000; Abcam) for 2 h at room temperature. The bands were visualized by ECL detection (Thermo Scientific), and all the experiments were repeated triple times.
Hrp conjugated goat anti mouse secondary antibody
Manufactured by Abcam
Sourced in United States
HRP-conjugated goat anti-mouse secondary antibody is a laboratory reagent used to detect and visualize mouse primary antibodies in various immunoassays and immunohistochemical applications. The antibody is conjugated to horseradish peroxidase (HRP), an enzyme that can catalyze a colorimetric reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit secondary antibody, by Abcam (6 mentions)
Bca kit, by Keygen Biotech (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab7959, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Keygen Biotech (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Luminal reagent, by Merck Group (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bbs 1, by Takara Bio (1 mentions)
Agarose gel dna purification kit version 2, by Takara Bio (1 mentions)
Bamhi, by Takara Bio (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Dojindo Laboratories (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Mini protean tgx 4 20 gel, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
19 protocols using hrp conjugated goat anti mouse secondary antibody
1
Western Blot Analysis of SNAP23 Protein
2
Western Blot Analysis of Cell Signaling
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Cells were harvested and treated with lysis buffer on ice (KeyGEN, Nanjing, China), and a BCA kit (KeyGEN, Nanjing, China) was used to quantify protein concentration. Equal amounts of protein were loaded in SDS–PAGE gels. After separation in the gel, the protein was transferred on a PVDF membrane. Membranes were blocked in 2% BSA in TBS-T for 1 h, and then incubated overnight (4°C) with antibodies against CDCA2 (Abcam, ab209656 1:1000), p21 (santa cruz, sc-397 1:500), p27 (santa cruz, sc-528 1:200), cyclin D1 (CST, 2978 1:1000), cyclin E1 (Abcam, ab7959 1:200) or β-actin (Cell Signaling, 8H10D10 1:1000). After being washed in TBS-T, membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:10,000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1:10,000; Abcam) for 2 h at room temperature. The blots were visualized by ECL detection (Thermo Scientific). All experiments were repeated at least three times independently.
3
Western Blot Analysis of E2F8, Cyclin D1, and β-Actin
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Cells were harvested and treated with lysis buffer on ice (KeyGEN, Nanjing, China), and a BCA kit (KeyGEN, Nanjing, China) was used to quantify protein concentration. Equal amounts of protein were loaded in SDS–PAGE gels. After separation in the gel, the protein was transferred on a PVDF membrane. Membranes were blocked in 2% BSA in TBS-T for 1 h, and then incubated overnight (4 °C) with antibodies against E2F8 (Abcam, ab185727 1:1000), cyclin D1 (CST, 2978 1:1000) or β-actin (Cell Signaling, 8H10D10 1:1000). After being washed in TBS-T, membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:10,000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1:10,000; Abcam) for 2 h at room temperature. The blots were visualized by ECL detection (Thermo Scientific). All experiments were repeated at least three times independently.
4
Protein Expression Analysis of Co-Cultured Cells
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Cells at 6 h and 12 h of co-culturing were lysed in RIPA buffer (Beyotime, Cat# P0013C, China). Proteins were separated by 10% SDS-PAGE and transferred to a methanol-activated PVDF membrane (Millipore, Cat# IPVH00010, USA).The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with p38 antibody (ImmunoWay, Cat# B6201, USA), JNK antibody (ImmunoWay, Cat# B4001,USA), ERK1/2 antibody (Cat# ImmunoWay, B2601, USA), cyclooxygenase-2 (COX-2) antibody (ImmunoWay, Cat#B7301, USA), phosphorylated p38 antibody (ImmunoWay, Cat# B4401, USA), phosphorylated JNK antibody (ImmunoWay, Cat# B6701,USA), phosphorylated ERK1/2 antibody (Immunollay, Cat#B0001, USA), and GAPDH antibody (Abcam, Cat# ab9485, USA) for 2 h. After 1 h incubation with goat-anti-mouse HRP-conjugated secondary antibody (Abcam, Cat# ab97051, USA), the protein bands were detected with luminal reagent (Millipore, Cat# WBKLS0500, USA). Their relative intensities were quantified using Adobe Photoshop software (Adobe Systems Inc., San Jose, USA).
5
Protein Expression Analysis in Cells
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Whole cells were homogenized and treated with lysis buffer on ice (Kaiji, Nanjing, China), and a BCA kit (Kaiji, Nanjing, China) was used to quantify protein concentrations. Equal amounts of protein were loaded in SDS–PAGE gels. After separation in the gel, the protein was transferred onto a PVDF membrane. The membranes were blocked in 2% BSA in TBST for 1 h, and incubated overnight (4°C) with antibodies against ZYG11A (Abcam, ab177696 1:1000), p21 (santa cruz, sc-397 1:500), p27 (santa cruz, sc-528 1:200), Cyclin D1 (CST, 2978 1:1000), Cyclin E1 (abcam, ab7959 1:200) or beta-actin (Cell Signaling, 8H10D10 1:1000). After washing in TBST, the membrane was incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:10,000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1:10,000; Abcam) for 2 h at room temperature. The blots were visualized by ECL detection (Thermo Scientific), and all experiments were repeated triple times.
6
Western Blot Analysis of EMT Markers
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Cells were harvested and treated with lysis buffer on ice, and then, the protein was quantified with a BCA kit. The procedures of western blot were complied with standard protocols as previously reported [15 (link)]. Antibodies against GLI3 (1 : 1000, Abcam, Cat. No. ab6050), GAPDH (1 : 1000, Abcam, Cat. No. ab8245), VIM (1 : 500, Abcam, Cat. No. ab92547), ZEB1 (1 : 1000, Abcam, Cat. No. ab203829), and CDH2 (1 : 500, Abcam, Cat. No. ab202030) were used during overnight incubation. After being washed in TBS-T, membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1 : 10,000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1 : 10,000; Abcam) for 2 h at room temperature. The blots were visualized by the ECL detection (Thermo Scientific). All experiments were repeated in triplicate.
7
Plasmid Construction and Cell Transfection
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The pGPU6/GFP/Neo expression vector was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The enzymes (T4 DNA ligase, BamH I, Bbs I, and Pst I enzymes), Escherichia coli JM109 cells, and Agarose Gel DNA Purification Kit Version 2.0 were purchased form Takara Biomedical Technology Co., Ltd. (Beijing, China). The plasmid kit was purchased form Sigma Co., LLC. (Shanghai, China). The Lipofectamine 2000 and TRIzol reagent were purchased from Invitrogen Applied Biosystems (Shanghai, China). The primary antibodies (HIF-2α and P-gp) and the goat anti-mouse HRP-conjugated secondary antibody were purchased from Abcam (Shanghai, China). MTT was purchased form Dojindo Laboratories (Kumamoto, Japan).
8
EV Protein Characterization by Western Blot
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CRL-5908 and CCL-185 cells were collected and washed with PBS in a 350 g × 5′ centrifugation. Previously isolated EVs and cells from both cell lines were lysed ice-cold Cell Lysis Buffer (Cell Signaling Technology. Ref 9803 S) according to manufacturer recommendations. Proteins were quantified with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific. Ref 23225) in Infinite® 200 PRO NanoQuant (TECAN). Thirty μg of protein from each sample were run in Mini-PROTEAN TGX 4–20% gels (Bio-Rad. Ref 561094) and transferred to nitrocellulose membranes (Bio-Rad. 162-0115). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline +1% Tween20 and incubated with primary antibodies, mouse monoclonal anti-CD9 (Thermo Fisher Scientific. Ref 10626D), mouse monoclonal anti-Hsp70 (BD Biosciences. Ref 554243), and mouse monoclonal anti-GM130 (BD Biosciences. Ref 610822), at 1:1000 dilution overnight at 4 °C. Then, membranes were washed with Tris-buffered saline +1% Tween20 and incubated with Goat anti-mouse HRP-conjugated secondary antibody (Abcam. Ref ab97023) at 1:5000 for 1 hour at room temperature. Finally, they were revealed using Clarity™ Western ECL Substrate (Bio-Rad. Ref 1705061) and pictures were taken in the ImageQuant LAS 4000 (GE HealthCare Life Sciences).
9
Western Blot Analysis of Cell Cycle Regulators
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Cells were harvested and treated with lysis buffer on ice (KeyGen Biotech. Co., Ltd.), and a bicinchoninic acid assay (BCA) kit (KeyGen Biotech. Co., Ltd.) was used to quantify protein concentration. Equal amounts of protein were loaded in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. After separation in the gel, the protein was transferred on a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in 2% bovine serum albumin (BSA) in Tris buffered saline with Tween 20 (TBS-T) for 1 h and then incubated overnight (4°C) with antibodies against CDC45 (sc-20685, 1:500; Santa Cruz Biotechnology), cyclin D1 (2978, 1:1000; Cell Signaling Technology), cyclin E1 (ab7959, 1:200; Abcam), CCNE2 (11935-1-AP, 1:500; Proteintech), p21 (sc-397, 1:500; Santa Cruz Biotechnology), p27 (sc-528, 1:200; Santa Cruz Biotechnology), or β-actin (8H10D10, 1:1000; Cell Signaling Technology). After being washed in TBS-T, membranes were incubated with goat antirabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; Abcam) or goat anti-mouse HRPconjugated secondary antibody (1:10,000; Abcam) for 2 h at room temperature. The blots were visualized by enhanced chemiluminescence (ECL) detection (Thermo Fisher Scientific). All experiments were repeated at least three times independently.
10
Evaluation of EMT Markers in Pancreatic Cancer
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PANC-1 and Aspc-1 cells were treated with pWPXL-miR-148a or pWPXL-control for 24 h at 37°C, homogenized in lysate buffer containing protease-inhibitor and centrifuged at 6,000 × g at 4°C for 10 min. The supernatant of was used for analysis of the total protein concentration using bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). For detection, proteins (40 µg) were loaded and separated using 12% SDS-PAGE gels, transferred to nitrocellulose membranes and hybridized as previously described (30 (link)). Subsequent to blocking in 5% skimmed milk, membranes were probed with primary antibodies MEG-3 (1:500; 5122 Cell Signaling Technology, Inc.), E-cadherin (1:1,000; ab11512), Vimentin (1:1,000; ab92547), Snail2 (1:1,000; ab180714), β-catenin (1:1,000; ab32572), C-myc (1:1,000; ab32072), Cyclin D1 (1:1,000; ab134175) and β-actin (1:1,000; ab8226) and incubated for 1 h at 37°C, followed by incubation with HRP-conjugated goat anti-mouse secondary antibody (1:2,000; ab6785; all Abcam) for 24 h at 4°C. The blots were visualized using a chemiluminescence kit (Thermo Fisher Scientific, Inc.). Quantity of protein was analyzed using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.).
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