PC-9 and PC-9/GR cell lines were provided by Dr. Jin Kyung Roh (Asan Medical Center, Asan Medical Institute of Convergence Science and Technology). Cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin in a 5% CO2 incubator at 37°C. Anti-Orai1, ‒proliferating cell nuclear antigen (PCNA), goat anti-rabbit horseradish peroxidase (HRP) secondary antibody, and goat anti-mouse HRP secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-LC3B, -STIM1, -SERCA2, ‒poly(ADP-ribose) polymerase (PARP), ‒β-actin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Afatinib was purchased from Selleckchem (Houston, TX, USA), and cyclopiazonic acid (CPA) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Bafilomycin A and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Hrp goat anti mouse secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The HRP goat anti-mouse secondary antibody is a laboratory reagent used to detect and visualize target proteins in various immunoassays. It consists of a goat-derived antibody that specifically binds to mouse primary antibodies, conjugated with the enzyme Horseradish Peroxidase (HRP). This enzyme can catalyze a colorimetric reaction, enabling the visualization and quantification of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Osb00017w, by Thermo Fisher Scientific (2 mentions)
β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit hrp secondary antibody, by Thermo Fisher Scientific (2 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Bafilomycin a, by Merck Group (1 mentions)
Afatinib, by Selleck Chemicals (1 mentions)
Antibiotic antimycotic mixture, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Sp600125, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem media, by Thermo Fisher Scientific (1 mentions)
Trypan blue vital stain, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
7 protocols using hrp goat anti mouse secondary antibody
1
Regulation of Orai1-Mediated Cell Signaling
2
Apoptosis and Cell Cycle Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UA, SP600125, MTT, Tris, glycine, NaCl, SDS, BSA, β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM) media, fetal bovine serum (FBS), 0.4% trypan blue vital stain, and antibiotic-antimycotic mixture were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies against procaspase-3, cyclin D1, and c-MYC as well as goat anti-rabbit-horse radish peroxidase (HRP) conjugate and goat anti-mouse HRP secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3
Western Blot Analysis of Pancreatic Cancer Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins obtained from pancreatic cancer tissues and cultured cells were used for Western blot analysis. The protein concentration in cell extracts was measured using the BCA Protein Assay Kit (Beyotime). Equal amount of protein was loaded and separated by SDS–PAGE, and then transferred onto PVDF membranes (Millipore). The membrane was blocked with 5% (w/v) non-fat dried skimmed milk powder at room temperature, and incubated with primary antibody rabbit anti-NUF2 (1:1000; Abcam, #ab122962), rabbit anti-Cyclin B1 (1:1000; Abcam, #ab7957), rabbit anti-Cdc25A (1:1000; Abcam, #ab991), mouse anti-Cdc2 (1:1000; Cell Signaling, #9116), or rabbit anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 1:8000; Proteintech Group, Inc., #10494-1-AP). Then the membrane was washed and incubated with goat anti-rabbit HRP (horseradish peroxidase) secondary antibody (1:5000; Santa Cruz, #SC-2054) or goat anti-mouse HRP secondary antibody (1:5000; Santa Cruz, #SC-2005) as described in the manufacturer's instructions. Then the bands were visualized after incubation with chemiluminescence detection reagent (Pierce). The protein level of GAPDH was used as a loading control.
4
Western Blot Analysis of Antiestrogen Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 350,000 cells per well were treated with 100 nM antiestrogen for 24 h in serum-free medium in 12-well plates. Cells were lysed with RIPA buffer (#89901) supplemented with a cocktail of protease and phosphatase inhibitors (#78410), both from ThermoFisher Scientific. Whole protein extracts were separated on 10% SDS–PAGE TGX gels (#5671035) and transferred to nitrocellulose membranes (#1704159), both from BioRad. Blots were incubated with either 1:1000 dilution of D12 ERα (#sc-8005), or 1:2000 dilution of β-actin (#sc-47778) primary antibodies, followed by 1:2000 dilution of goat anti-mouse-HRP secondary antibody (#sc-2005) (all antibodies from Santa Cruz Biotechnology). Signal was detected with Super Signal Femto chemiluminescent reagent (ThermoFisher Scientific, #34095) and ERα bands normalized to β-actin to control for differences in loading. Imaging was performed on FluorChem Imager by Alpha Innotech. Data shown in figures are representative experiments reproduced at least three times in independent Western assays. An uncropped image of the immunoblot is shown in Supplementary Fig. 5 .
5
Immunohistochemical Analysis of Apoptosis-Inducing Factor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor sections were heat-treated to retrieve antigen, blocked in 3% BSA in PBST for 30 min and incubated with AIF antibody (Santa Cruz Biotech, sc-55519, USA; 1∶200 dilution in PBST) overnight at 4°C [28] (link). HRP-goat anti-mouse secondary antibody (1∶1000 dilution in PBS) (Santa Cruz, USA) was added for 1 h and detected by diaminobenzidine (Sigma, USA). Cells were counterstained with hematoxylin. Antibody testing is showed on Fig. S1 .
For western blot analysis, cryostat sliced frozen tissue were lysed in RIPA buffer with PMSF and protease inhibitor cocktails (Thermo, USA). Crude protein lysates were used for analysis. All the western blot tests were repeated at least three times. Quantity One software was used in the quantification of western blot data.
For western blot analysis, cryostat sliced frozen tissue were lysed in RIPA buffer with PMSF and protease inhibitor cocktails (Thermo, USA). Crude protein lysates were used for analysis. All the western blot tests were repeated at least three times. Quantity One software was used in the quantification of western blot data.
6
Western Blot Analysis of BDNF Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (Cyto Matin Gene Co., Iran) according to the manufacturer's instructions. 20 µg of total cell proteins were run in a 12% SDS-polyacrylamide gel and blotted to the PVDF membrane (Bio-Rad, USA). Membranes were incubated overnight at 4 °C with mature BDNF primary antibody (ThermoFisher OSB00017W, USA) followed by an hour incubation with HRP goat anti-rabbit secondary antibodies (Santa sc-2004, USA) at room temperature. After chemiluminescence visualization, reprobing procedure was done by 0.2 M NaOH for 5 minutes.
Next, beta-actin antibody (Santa Cruz sc-47778, USA) as the internal control was applied on the membrane at 4 °C overnight, followed by an hour incubation with HRP goat anti-mouse secondary antibody (Santa Cruz sc-2005, USA) at room temperature [28] . Blots were visualized by chemiluminescence (ECL; Cyto Matin Gene, Iran) and the obtained bands were quantified using ImageJ software.
Next, beta-actin antibody (Santa Cruz sc-47778, USA) as the internal control was applied on the membrane at 4 °C overnight, followed by an hour incubation with HRP goat anti-mouse secondary antibody (Santa Cruz sc-2005, USA) at room temperature [28] . Blots were visualized by chemiluminescence (ECL; Cyto Matin Gene, Iran) and the obtained bands were quantified using ImageJ software.
7
Western Blot Analysis of BDNF Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (Cyto Matin Gene Co., Iran) according to the manufacturer's instructions. 20 µg of total cell proteins were run in a 12% SDS-polyacrylamide gel and blotted to the PVDF membrane (Bio-Rad, USA). Membranes were incubated overnight at 4 °C with mature BDNF primary antibody (ThermoFisher OSB00017W, USA) followed by an hour incubation with HRP goat anti-rabbit secondary antibodies (Santa sc-2004, USA) at room temperature. After chemiluminescence visualization, reprobing procedure was done by 0.2 M NaOH for 5 minutes.
Next, beta-actin antibody (Santa Cruz sc-47778, USA) as the internal control was applied on the membrane at 4 °C overnight, followed by an hour incubation with HRP goat anti-mouse secondary antibody (Santa Cruz sc-2005, USA) at room temperature [28] . Blots were visualized by chemiluminescence (ECL; Cyto Matin Gene, Iran) and the obtained bands were quantified using ImageJ software.
Next, beta-actin antibody (Santa Cruz sc-47778, USA) as the internal control was applied on the membrane at 4 °C overnight, followed by an hour incubation with HRP goat anti-mouse secondary antibody (Santa Cruz sc-2005, USA) at room temperature [28] . Blots were visualized by chemiluminescence (ECL; Cyto Matin Gene, Iran) and the obtained bands were quantified using ImageJ software.
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!