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reviewed by: Anonymous
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ChIP |
Human Breast Cancer Cell Lines and Human |
05/17/2020 |
Summary
| Application | Chromatin Immunoprecipitation | | Sample Tested | Breast cancer cells | | Species | Human Breast Cancer Cell Lines and Human | | Lot | AN-1 |
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reviewed by: Anonymous
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WB |
Human |
04/16/2019 |
Summary
| Application | Western Blot | | Sample Tested | 16-HBE | | Species | Human | | Lot | AL-6 |
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reviewed by: Anonymous
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WB |
Human |
09/18/2018 |
Summary
| Application | Western Blot | | Sample Tested | Human Cancer Cells | | Species | Human | | Lot | AN-1 |
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reviewed by: Anonymous
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IHC-P |
Mouse |
10/25/2017 |
Summary
| Application | Immunohistochemistry-Paraffin | | Sample Tested | IHC on parrafin sections (mouse) | | Species | Mouse | | Lot | AK-4 |
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reviewed by: Kristina Cook
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WB |
Human |
02/23/2017 |
Summary
| Application | Western Blot | | Sample Tested | U251 glioma cell line; whole cell lysate | | Species | Human |
Comments
| Comments | 4 hour hypoxia (0.5% oxygen) incubation |
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reviewed by: Anonymous
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WB |
Mouse |
09/21/2016 |
Summary
| Application | Western Blot | | Sample Tested | Mouse kidney | | Species | Mouse |
Blocking
| Blocking Details | Blocked with 5% Nonfat Dry Milk in 1x TBS-T, 1 hour at room temperature |
Primary Anitbody
| Dilution Ratio | Dilution - 5% BSA in TBS-T solution buffer |
Secondary Antibody
| Secondary Description | Horse Anti-mouse IgG, HRP-linked Antibody | | Secondary Manufacturer Cat# | #7076 | | Secondary Concentration | 2ul in 10ml of 5% Milk in TBS-T |
Details
| Detection Notes | Chemiluminescent detection |
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reviewed by: Anonymous
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WB |
Mouse |
08/06/2016 |
Summary
| Application | Western Blot | | Species | Mouse | | File | View PDF |
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reviewed by: Zhongxiao Wang
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WB |
Mouse |
10/05/2015 |
Summary
| Application | Western Blot | | Sample Tested | mouse retina | | Species | Mouse | | Lot | NB100-479 |
Blocking
| Blocking Details | 5%milk/T-BST buffer, room temparature, 2hr |
Primary Anitbody
| Dilution Ratio | 1:1000 in 5%milk/T-BST buffer, 4 degree, overnight |
Secondary Antibody
| Secondary Description | Goat anti-rabbit IgG, HRP | | Secondary Manufacturer Cat# | Sigma AQ132P | | Secondary Concentration | 1:3000 |
Details
| Detection Notes | Develop machine, 30s exposure shows clear bands |
Comments
| Comments | There's a non-specific band above for mouse retina samples |
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reviewed by: Anonymous
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WB |
Human |
08/23/2015 |
Summary
| Application | Western Blot | | Sample Tested | Human cancer cell whole cell lysate | | Species | Human |
Blocking
| Blocking Details | Blocking with 5% non-fat milk for 30 min |
Primary Anitbody
| Dilution Ratio | 1:1000, overnight |
Secondary Antibody
| Secondary Description | Donkey anti-rabbit IgG, HRP | | Secondary Manufacturer Cat# | GE Healthcare, NA934V | | Secondary Concentration | 1:2000 |
Details
| Detection Notes | Regular Protocol |
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reviewed by: Anonymous
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WB |
Human |
03/27/2015 |
Summary
| Application | Western Blot | | Sample Tested | Hela lysates treated with MG132 | | Species | Human |
Blocking
| Blocking Details | 5% milk in TBST |
Primary Anitbody
Secondary Antibody
| Secondary Description | Goat anti-rabbit |
Details
| Detection Notes | using the regular pico from pierece, detection by Bio-rad imager, 1 min exposure time, DMSO as the NC, MG132 as the PC |
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reviewed by: Anonymous
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WB |
Mouse |
02/18/2015 |
Summary
| Application | Western Blot | | Sample Tested | Mouse heart | | Species | Mouse | | Lot | J1 |
Blocking
| Blocking Details | 5% milk in TBST for 1h at RT |
Primary Anitbody
| Dilution Ratio | 1:1000 in 5% milk TBST overnight at 4°C |
Secondary Antibody
| Secondary Description | Goat anti-rabbit IgG, HRP-coupled, 1:10 000 in 5% milk TBST, 1h at RT | | Secondary Manufacturer Cat# | AffiniPure Goat Anti-Rabbit IgG (H+L) from Jackson Immunolab | | Secondary Concentration | 1:10 000 |
Details
| Detection Notes | ECL Clarity, exposure time 30s |
Comments
| Comments | - The signal was strong, it might be useful to decrease primary antibody concentration to reduce background noise. |
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reviewed by: Anonymous
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ChIP |
Human |
12/15/2014 |
Summary
| Application | Chromatin Immunoprecipitation | | Sample Tested | HEK293 | | Species | Human | | Lot | AA-1 |
Primary Anitbody
| Dilution Ratio | Use 5ug antibody per 1ml lysate, and incubate at 4oC overnight |
Details
| Detection Notes | Real-time PCR. | | Fixation/Cross-Linking | Elute the beads with 1% SDS, 0.1M NaHCO3 and then reverse the cross-linking by incubating at 65oC for 6 hours. | | DNS Sharing | Covaris sonicator, 6 seconds for 200-500bp | | Immunoprecipitation Details | Millipore protein G magnetic beads 50ul/1ml lysate at room temperature for 10 minutes. | | Crosslinking/Reversal Details | 1% formaldehyde for 5 minutes at room temperature | | DNA Prurification Details | Qiagen PCR cleanup kit. |
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reviewed by: Anonymous
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WB |
Mouse |
12/12/2014 |
Summary
| Application | Western Blot | | Sample Tested | See PMID 23232625 | | Species | Mouse |
Blocking
| Blocking Details | See PMID 23232625 |
Details
| Detection Notes | See PMID 23232625 |
Comments
| Comments | This antibody against HIF-1alpha reproducibly recognizes a single band in western blots assessing protein lysates from hypoxia-treated primary mouse aortic endothelial cells.Published in PMID: 23232625. |
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reviewed by: Xiuquan Luo
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WB |
Human |
09/12/2014 |
Summary
| Application | Western Blot | | Sample Tested | HEK293 cell lysate | | Species | Human | | Comments | Antibody detects some non-specific bands, but give a good HIF1a signal at indicated MW. |
Blocking
| Blocking Details | 1xPBST with 5% non-fat milk, RT 1 hour |
Primary Anitbody
| Dilution Ratio | 1xPBST with 5% non-fat milk, overnight 4C, 1:1000 dilution |
Secondary Antibody
| Secondary Description | Goat anti-rabbit IgG-HRP | | Secondary Manufacturer Cat# | sc-2004 | | Secondary Concentration | 1:2000 dilution |
Details
| Detection Notes | ECL dura, exposured for 2-5 min |
Comments
| Comments | Antibody detects some non-specific bands, but give a good HIF1a signal at indicated MW. |
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reviewed by: Ming Lu
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WB |
Human |
08/15/2014 |
Summary
| Application | Western Blot | | Sample Tested | Human Pancreatic Cancer Cell line | | Species | Human | | Lot | AA | | Comments | Very clean antibody, but it has multiple bands. |
Blocking
| Blocking Details | 2% cold fish geletin in TBS buffer, room temperature for 1hour |
Primary Anitbody
| Dilution Ratio | 1:1000 overnight at 4C in blocking buffer |
Secondary Antibody
| Secondary Description | Goat anti-rabbit IgG, HRP | | Secondary Manufacturer Cat# | Cell Signaling, #7074 | | Secondary Concentration | 1:5000 |
Details
| Detection Notes | 1 min exposure shows band at correct MW. |
Comments
| Comments | Very clean antibody, but it has multiple bands. |
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reviewed by: Molly Jenkins
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WB |
Human |
07/18/2014 |
Summary
| Application | Western Blot | | Sample Tested | Nuclear fraction, Human cell line | | Species | Human |
Blocking
| Blocking Details | 5% milk in TBST, 1hr at RT |
Primary Anitbody
Secondary Antibody
| Secondary Description | Goat anti-rabbit HRP | | Secondary Concentration | 1:20,000 |
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reviewed by: Jason Lee
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WB |
Human |
10/29/2013 |
Summary
| Application | Western Blot | | Sample Tested | Human MCF7 breast epithelial cells | | Species | Human | | Lot | Lot X-1 | | Comments | nuclear extract resolved in 8% SDS-PAGE gel |
Blocking
| Blocking Details | 3% BSA in TBST buffer 1 hour room temp |
Primary Anitbody
| Dilution Ratio | 1:2000 dilution in 3% BSA TBST 1 hour room temp |
Secondary Antibody
| Secondary Description | goat anti-rabbit IgG HRP | | Secondary Manufacturer Cat# | Sigma | | Secondary Concentration | 1:5000 in 5% skim milk TBST |
Details
| Detection Notes | LAS4000 2 second exposure |
Comments
| Comments | nuclear extract resolved in 8% SDS-PAGE gel |
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reviewed by: Anonymous
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IHC-P |
Mouse |
06/27/2013 |
Summary
| Application | Immunohistochemistry-Paraffin | | Sample Tested | paraffin section of skin wound | | Species | Mouse | | Comments | Antibody works much better when you find the necessary dilution which is quite low. Works best around 1:5000 to 1:7000. |
Primary Anitbody
| Dilution Ratio | 1:6000, room tempereature in Dako antibody diluent, overnight, 4oC |
Secondary Antibody
| Secondary Description | anti rabbit HRP polymer, Dako |
Details
| Fixation Details | 4% paraformaldehyde, DAKO heat mediated antigen retrieval, 20 mins, 98 deg C | | Wash Description | Dako wash buffer, Tris, contains Tween 20 |
Comments
| Comments | Antibody works much better when you find the necessary dilution which is quite low. Works best around 1:5000 to 1:7000. |
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reviewed by: Sandhya Khurana
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WB |
Rat |
02/05/2012 |
Summary
| Application | Western Blot | | Sample Tested | PC12 nuclear extract, Sample Amount: 50ug | | Species | Rat |
Blocking
| Blocking Details | Blocking Buffer: 5% Milk in TBST, Blocking Time: 30 minutes, Blocking Temp: Room temperature |
Primary Anitbody
| Dilution Ratio | Dilution Ratio: 1:1000, Incubation Dilution Buffer: 5% Milk/TBST, Incubation Time: overnight, Incubation Temp: 4 C |
Secondary Antibody
| Secondary Description | Secondary Ab Dilution Ratio: 1:2000 |
Details
| Detection Notes | Method:ECL, Time:2min, PosCtrl: Lysate from cells overexpressing HIF1a, NegCtrl: Untreated cells, Specific Bands:110-120kDa |
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reviewed by: Anonymous
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WB |
Rat |
01/03/2012 |
Summary
| Application | Western Blot | | Sample Tested | PC12 cell whole lysate, Sample Amount: 25ug | | Species | Rat | | Lot | P1 | | Comments | This antibody detected HIF-1a. |
Blocking
| Blocking Details | Blocking Buffer: 5% Milk in TBST, Blocking Time: 1 hour, Blocking Temp: Room temperature |
Primary Anitbody
| Dilution Ratio | Dilution Ratio: 1:1000, Incubation Dilution Buffer: blocking buffer, Incubation Time: overnight, Incubation Temp: 4 C |
Comments
| Comments | This antibody detected HIF-1a. |
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reviewed by: Anonymous
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IHC-P |
Mouse |
07/30/2011 |
Summary
| Application | Immunohistochemistry-Paraffin | | Sample Tested | normal mouse kidney | | Species | Mouse | | Comments | Nuclear staining of (active) Hif-1alpha + cytoplasmic staining of (inactive) Hif-1alpha in normal mouse kidney (formalin-fixed, paraffin-embedded). No staining detected in controls. Staining done according to CSAII kit instructions. In addition, a permeabilization step was included (20 min. PBS + 0.5% Triton X-100) prior to Protein Block. Detection Method: DAB, Detection Time: 10 minutes, Positive Control: normal mouse kidney, Negative Control: rabbit isotype, antibody pre-absorbed with Hif-1alpha protein (NBC1-18422), Deparaffinization: 2x5 min. xylene, Antigen Retrieval: Target Retrieval Solution (Dako), 25 min. in pressure cooker |
Blocking
| Blocking Details | Blocking Buffer: Protein Block (CSAII kit, Dako), Blocking Time: 15 minutes, Blocking Temp: room temperature |
Primary Anitbody
| Dilution Ratio | Dilution Ratio: 1:20,000, Incubation Dilution Buffer:Ab Diluent, background reducing (Dako), Incubation Time/Temp:overnight @ 4C |
Secondary Antibody
| Secondary Description | Secondary Ab: CSAII Rabbit Link (Dako), Secondary Ab Dilution: 1:1 | | Secondary Manufacturer Cat# | K1501 |
Details
| Detection Notes | Please see Comments | | Fixation Details | Preparation: perfused PBS-formalin, postfixed overnight @ 4C in formalin, paraffin-embedded, Deparaffinization: 2x5 min. xylene |
Comments
| Comments | Nuclear staining of (active) Hif-1alpha + cytoplasmic staining of (inactive) Hif-1alpha in normal mouse kidney (formalin-fixed, paraffin-embedded). No staining detected in controls. Staining done according to CSAII kit instructions. In addition, a permeabilization step was included (20 min. PBS + 0.5% Triton X-100) prior to Protein Block. Detection Method: DAB, Detection Time: 10 minutes, Positive Control: normal mouse kidney, Negative Control: rabbit isotype, antibody pre-absorbed with Hif-1alpha protein (NBC1-18422), Deparaffinization: 2x5 min. xylene, Antigen Retrieval: Target Retrieval Solution (Dako), 25 min. in pressure cooker |
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reviewed by: Paramahamsa Maturu
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IHC-P |
Human |
03/01/2011 |
Summary
| Application | Immunohistochemistry-Paraffin | | Sample Tested | Human Kidney tumor | | Species | Human | | Lot | I | | Comments | That works great without any background staining in the negative control. Detection Method: ABC and DAB, Positive Control: Glioblastoma, Negative Control: Without primary Ab, Deparaffinization: Xylen and ethanol, Antigen Retrieval: Tris -EDTA pH 9.0, 45 minutes at 95 degrees Celsius in water bath |
Blocking
| Blocking Details | Blocking Buffer:.10% Normal Goat Serum in 1XPBS, Blocking Time: 1 hour, Blocking Temp: RT in humidified chamber |
Primary Anitbody
| Dilution Ratio | Dilution Ratio: 1:100, Incubation Dilution Buffer: 2.5% Normal Goat Serum in 1X PBS, Incubation Time/Temp: overnight at 4C |
Secondary Antibody
| Secondary Description | Secondary Ab: Biotinylated Anti Rabbit IgG (H+L) made in Goat, Secondary Ab Dilution: 1:500 | | Secondary Manufacturer Cat# | BA-1000 |
Details
| Detection Notes | Please see Comments | | Fixation Details | Sample Preparation: Paraffin embedded, Deparaffinization: Xylen and ethanol |
Comments
| Comments | That works great without any background staining in the negative control. Detection Method: ABC and DAB, Positive Control: Glioblastoma, Negative Control: Without primary Ab, Deparaffinization: Xylen and ethanol, Antigen Retrieval: Tris -EDTA pH 9.0, 45 minutes at 95 degrees Celsius in water bath |
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reviewed by: JORCYK LAB
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WB |
Mouse |
02/01/2011 |
Summary
| Application | Western Blot | | Sample Tested | 4T1 cells whole cell lysate | | Species | Mouse | | Comments | The Hif1a band shows up even if the lysate concentration is very low. There are some background bands, but is acceptable, and there is quite a bit of lot to lot variations with this product... unfortunately. |
Blocking
| Blocking Details | Blocking Buffer: 5% milk or 3% BSA, Blocking Time: overnight, Blocking Temp: 4 degrees Celsius |
Primary Anitbody
| Dilution Ratio | Dilution Ratio: 1:1000-1:5000, Incubation Dilution Buffer: TBS-T or PBS-T, Incubation Time: 1 hr-overnight, Incubation Temp: 4C |
Secondary Antibody
| Secondary Description | Secondary Ab Dilution Ratio: 1:10,000-1:150,000 |
Details
| Detection Notes | Method: ECL, Exposure Time: 10sec-10min, Pos.Ctrl: ECL reactive ladder, Bands:~120kDa(specific), ~80,55,72kDa(non-specific) |
Comments
| Comments | The Hif1a band shows up even if the lysate concentration is very low. There are some background bands, but is acceptable, and there is quite a bit of lot to lot variations with this product... unfortunately. |
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