Product Page for RHOT2 Antibody (ARP62299_P050)
Researcher: Jin-Mi Heo
Application: Western Blotting
Species+tissue/cell type: Lane 1: 20ug untransfected HEK293T Lane 2: 20ug RHOT2 transfected HEK293T
Primary antibody dilution:1:1000
Secondary antibody: Anti-rabbit-HRP
Secondary antibody dilution: 1:2000
Product Page for CAV2 antibody – N-terminal region (AVARP09020_P050)
Researcher: Dr. Hiten D. Mistry and Anna Czajka, King’s College London; Lesia Kurlak, University of Nottingham
Application: IHC
Species+tissue/cell type: Human placental tissue
Primary antibody dilution:1:50
Secondary antibody: Goat anti rabbit-HRP
Secondary antibody dilution: 1:10,000
| Questionnaire: | |
| How do Aviva’s reagents play a role in your experimental goals? | It is one of our main antibodies of interest. |
| How would you rate this antibody on a scale from 1-5 (5=best) and why? | 4 |
| Would you use this antibody in future experiments? | Yes |
| Have you used another antibody which has worked in your application? | No |
| Do you believe the information about the reagent on Aviva’s website is correct? | Yes |
| If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? | Yes |
| How did you store the antibody after re-suspension? | -20 d C |
| Sample Description (please include species type and tissue/cell type): | Human Placental tissues samples, paraffin-embedded slides. |
| Please explain fixation solution/method used (formalin, periodate-lysine-paraformaldehyde, acetone, etc.)? | Formalin fixed |
| How many different experimental trials were conducted using the antibody sample? | 6 |
| Primary antibody dilution, incubation time and temperature: | 1 in 200 dilution |
| Secondary antibody used, dilution, incubation time and temperature: | 1 in 10000 goat anti rabbit, 30 minutes at room temperature |
| From your IHC/ICC images, briefly explain the colors of each stain and counterstain: | DAB staining and counter stained with haemotoxylin |
| Did you use an antigen retrieval method? If so, please explain? | Heat-induced antigen retrieval method. |
| What controls were used in your experiment? | Negative control: IgG rabbit negative. Positive control: Skeletal muscle. |
| Please include your detailed tissue preparation and staining procedure/protocol here: | Mount paraffin-embedded sections on APES-coated slides. Dry the sections overnight 37 d C. 1. Dewax sections in xylene baths (2x5mins) 2. Immerse in absolute alcohol (IMS is cheaper) (2×5 mins) 3. Immerse in fresh 0.5% H2O2 in methanol-peroxidase block (store up to max 1wk at 4 d C) (10 mins) 4. Immerse in alcohol (2×5 mins) 5. Immerse in 70% alcohol (5 mins) 6. Wash in runnig water (5 mins) 7. Microwave for 15 mins at medium setting in Citrate buffer solution (Heat induced epitope retieval). Time depends on equipment [20 mins on high power] secondary Ab normal serum (dil 1/10 in TBS/BSA) (30 mins) assessed in a validation run (30 mins) |
Product Page for Sdpr antibody – N-terminal region (ARP50499_P050)
Researcher: Dr. Hiten D. Mistry and Anna Czajka, King’s College London; Lesia Kurlak, University of Nottingham
Application: IHC
Species+tissue/cell type: Human placental tissue
Primary antibody dilution:1:50
Secondary antibody: Goat anti rabbit-HRP
Secondary antibody dilution: 1:10,000
| Questionnaire: | |
| How do Aviva’s reagents play a role in your experimental goals? | It is one of our main antibodies of interest. |
| How would you rate this antibody on a scale from 1-5 (5=best) and why? | 4 |
| Would you use this antibody in future experiments? | Yes |
| Have you used another antibody which has worked in your application? | No |
| Do you believe the information about the reagent on Aviva’s website is correct? | Yes |
| If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? | Yes |
| How did you store the antibody after re-suspension? | -20 d C |
| Sample Description (please include species type and tissue/cell type): | Human Placental tissues samples, paraffin-embedded slides. |
| Please explain fixation solution/method used (formalin, periodate-lysine-paraformaldehyde, acetone, etc.)? | Formalin fixed |
| How many different experimental trials were conducted using the antibody sample? | 6 |
| Primary antibody dilution, incubation time and temperature: | 1 in 50 dilution |
| Secondary antibody used, dilution, incubation time and temperature: | 1 in 10000 goat anti rabbit, 30 minutes at room temperature |
| From your IHC/ICC images, briefly explain the colors of each stain and counterstain: | DAB staining and counter stained with haemotoxylin |
| Did you use an antigen retrieval method? If so, please explain? | Heat-induced antigen retrieval method. |
| What controls were used in your experiment? | Negative control: IgG rabbit negative. Positive control: Skeletal muscle. |
| Please include your detailed tissue preparation and staining procedure/protocol here: | Mount paraffin-embedded sections on APES-coated slides. Dry the sections overnight 37 d C. 1. Dewax sections in xylene baths (2x5mins) 2. Immerse in absolute alcohol (IMS is cheaper) (2×5 mins) 3. Immerse in fresh 0.5% H2O2 in methanol-peroxidase block (store up to max 1wk at 4 d C) (10 mins) 4. Immerse in alcohol (2×5 mins) 5. Immerse in 70% alcohol (5 mins) 6. Wash in runnig water (5 mins) 7. Microwave for 15 mins at medium setting in Citrate buffer solution (Heat induced epitope retieval). Time depends on equipment [20 mins on high power] secondary Ab normal serum (dil 1/10 in TBS/BSA) (30 mins) assessed in a validation run (30 mins) |
Product Page for NOS2 antibody – N-terminal region (ARP63000_P050)
Researcher: Dr. Hiten D. Mistry and Anna Czajka, King’s College London; Lesia Kurlak, University of Nottingham
Application: IHC
Species+tissue/cell type: Human placental tissue
Primary antibody dilution:1:50
Secondary antibody: Goat anti rabbit-HRP
Secondary antibody dilution: 1:10,000
| Questionnaire: | |
| How do Aviva’s reagents play a role in your experimental goals? | It is one of our main antibodies of interest. |
| How would you rate this antibody on a scale from 1-5 (5=best) and why? | 4 |
| Would you use this antibody in future experiments? | Yes |
| Have you used another antibody which has worked in your application? | No |
| Do you believe the information about the reagent on Aviva’s website is correct? | Yes |
| If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? | Yes |
| How did you store the antibody after re-suspension? | -20 d C |
| Sample Description (please include species type and tissue/cell type): | Human Placental tissues samples, paraffin-embedded slides. |
| Please explain fixation solution/method used (formalin, periodate-lysine-paraformaldehyde, acetone, etc.)? | Formalin fixed |
| How many different experimental trials were conducted using the antibody sample? | 6 |
| Primary antibody dilution, incubation time and temperature: | 1 in 50 dilution |
| Secondary antibody used, dilution, incubation time and temperature: | 1 in 10000 goat anti rabbit, 30 minutes at room temperature |
| From your IHC/ICC images, briefly explain the colors of each stain and counterstain: | DAB staining and counter stained with haemotoxylin |
| Did you use an antigen retrieval method? If so, please explain? | Heat-induced antigen retrieval method. |
| What controls were used in your experiment? | Negative control: IgG rabbit negative. Positive control: Skeletal muscle. |
| Please include your detailed tissue preparation and staining procedure/protocol here: | Mount paraffin-embedded sections on APES-coated slides. Dry the sections overnight 37 d C. 1. Dewax sections in xylene baths (2x5mins) 2. Immerse in absolute alcohol (IMS is cheaper) (2×5 mins) 3. Immerse in fresh 0.5% H2O2 in methanol-peroxidase block (store up to max 1wk at 4 d C) (10 mins) 4. Immerse in alcohol (2×5 mins) 5. Immerse in 70% alcohol (5 mins) 6. Wash in runnig water (5 mins) 7. Microwave for 15 mins at medium setting in Citrate buffer solution (Heat induced epitope retieval). Time depends on equipment [20 mins on high power] secondary Ab normal serum (dil 1/10 in TBS/BSA) (30 mins) assessed in a validation run (30 mins) |
Product Page for PDE5A antibody – C-terminal region (ARP63250_P050)
Researcher: Dr. Hiten D. Mistry and Anna Czajka, King’s College London; Lesia Kurlak, University of Nottingham
Application: IHC
Species+tissue/cell type: Human placental tissue
Primary antibody dilution:1:100
Secondary antibody: Goat anti rabbit-HRP
Secondary antibody dilution: 1:10,000
| Questionnaire: | |
| How do Aviva’s reagents play a role in your experimental goals? | It is one of our main antibodies of interest. |
| How would you rate this antibody on a scale from 1-5 (5=best) and why? | 4 |
| Would you use this antibody in future experiments? | Yes |
| Have you used another antibody which has worked in your application? | No |
| Do you believe the information about the reagent on Aviva’s website is correct? | Yes |
| If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? | Yes |
| How did you store the antibody after re-suspension? | -20 d C |
| Sample Description (please include species type and tissue/cell type): | Human Placental tissues samples, paraffin-embedded slides. |
| Please explain fixation solution/method used (formalin, periodate-lysine-paraformaldehyde, acetone, etc.)? | Formalin fixed |
| How many different experimental trials were conducted using the antibody sample? | 6 |
| Primary antibody dilution, incubation time and temperature: | 1 in 100 dilution |
| Secondary antibody used, dilution, incubation time and temperature: | 1 in 10000 goat anti rabbit, 30 minutes at room temperature |
| From your IHC/ICC images, briefly explain the colors of each stain and counterstain: | DAB staining and counter stained with haemotoxylin |
| Did you use an antigen retrieval method? If so, please explain? | Heat-induced antigen retrieval method. |
| What controls were used in your experiment? | Negative control: IgG rabbit negative. Positive control: Skeletal muscle. |
| Please include your detailed tissue preparation and staining procedure/protocol here: | Mount paraffin-embedded sections on APES-coated slides. Dry the sections overnight 37 d C. 1. Dewax sections in xylene baths (2x5mins) 2. Immerse in absolute alcohol (IMS is cheaper) (2×5 mins) 3. Immerse in fresh 0.5% H2O2 in methanol-peroxidase block (store up to max 1wk at 4 d C) (10 mins) 4. Immerse in alcohol (2×5 mins) 5. Immerse in 70% alcohol (5 mins) 6. Wash in runnig water (5 mins) 7. Microwave for 15 mins at medium setting in Citrate buffer solution (Heat induced epitope retieval). Time depends on equipment [20 mins on high power] secondary Ab normal serum (dil 1/10 in TBS/BSA) (30 mins) assessed in a validation run (30 mins) |
Product Page for RUVBL1 antibody – N-terminal region (ARP32349_P050)
Researcher: Sustackova Gabriela
Application: Western Blotting
Species+tissue/cell type: Lane 1: 30ug K562 lysate
Primary antibody dilution:1:200
Secondary antibody: Anti-rabbit-HRP
Secondary antibody dilution: 1:1000
| Questionnaire: | |
| How do Aviva’s reagents play a role in your experimental goals? | I wanted to find antibody which would work for Drosophila cells |
| Have you used another antibody which has worked in your application? | Yes |
| Do you believe the information about the reagent on Aviva’s website is correct? | Yes |
| How did you store the antibody after re-suspension? | In aliquots at -20°C |
| Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel): | 1) Drosophila, 2) Drosophila S2 cells, 3) 30 μg |
| How many different experimental trials were conducted using the antibody sample? | 7 |
| How was this sample prepared? | Cells were lysed by sample buffer (50 mM Tris, 10% glycerol, 1% SDS), then sonicated and boiled with 2-mercaptoethanol and bromophenol blue, storage at -20°C |
| Primary antibody dilution and incubation time: | 1:200 in 5% milk, incubated either overnight at 4°C or 1h at room temperature |
| Secondary antibody used and dilution and incubation time: | Goat anti-rabbit conjugated with HRP, incubated for 1h at room temperature |
| What controls were used in your experiment (positive/negative)? | Positive control – K562 cells/ negative control – knockdown of gene in Drosophila cells using dsRNA |
| Please include your detailed WB Procedure/Protocol here: | Samples ran in 12% SDS-polyacrylamide gel at 100-130V. Proteins were transferred from gel to membrane in 1h 30min at 300mA. Blocking – 1h at room temperature in 5% milk. Incubation with RUVBL1 antibody – either overnight at 4°C or 1h at room temperature. Dilution 1:200 in 5% milk. Washing – 3x 10 minutes in PBST buffer (0.05%). Incubation with secondary antibody – 1h at room temperature. Dilution of goat-anti-rabbit – HRP antibody 1:1000 in 5% milk. Washing – 3x 10 minutes in PBST (0.05%). Developed by Clarity Western ECL substrate (Biorad) and exposed to film. comment to the image: line 1 – K562 positive control line 2 – S2 cells control line 3 – S2 cells with silenced RUVBL1 RUVBL1 should show band around 50 kDa, however in S2 cells there wasn’t any band about this size even with high concentration of antibody and loading of 30 μg of protein sample. In K562 leukemic cells there was a band above 55 kDa (MW marker PageRuler Plus Prestained Protein Ladder, Thermo Scientific). |
Product Page for RUVBL2 antibody – N-terminal region (ARP32379_P050)
Researcher: Sustackova Gabriela
Application: Western Blotting
Species+tissue/cell type: Lane 1: 30ug K562 lysate
Primary antibody dilution:1:200
Secondary antibody: Anti-rabbit-HRP
Secondary antibody dilution: 1:1000
| Questionnaire: | |
| How do Aviva’s reagents play a role in your experimental goals? | I wanted to find antibody which would work for Drosophila cells |
| Have you used another antibody which has worked in your application? | Yes |
| Do you believe the information about the reagent on Aviva’s website is correct? | Yes |
| How did you store the antibody after re-suspension? | In aliquots at -20°C |
| Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel): | 1) Drosophila, 2) Drosophila S2 cells, 3) 30 μg |
| How many different experimental trials were conducted using the antibody sample? | 7 |
| How was this sample prepared? | Cells were lysed by sample buffer (50 mM Tris, 10% glycerol, 1% SDS), then sonicated and boiled with 2-mercaptoethanol and bromophenol blue, storage at -20°C |
| Primary antibody dilution and incubation time: | 1:200 in 5% milk, incubated either overnight at 4°C or 1h at room temperature |
| Secondary antibody used and dilution and incubation time: | Goat anti-rabbit conjugated with HRP, incubated for 1h at room temperature |
| What controls were used in your experiment (positive/negative)? | Positive control – K562 cells/ negative control – knockdown of gene in Drosophila cells using dsRNA |
| Please include your detailed WB Procedure/Protocol here: | Samples ran in 12% SDS-polyacrylamide gel at 100-130V. Proteins were transferred from gel to membrane in 1h 30min at 300mA. Blocking – 1h at room temperature in 5% milk. Incubation with RUVBL2 antibody – either overnight at 4°C or 1h at room temperature. Dilution 1:200 in 5% milk. Washing – 3x 10 minutes in PBST buffer (0.05%). Incubation with secondary antibody – 1h at room temperature. Dilution of goat-anti-rabbit – HRP antibody 1:1000 in 5% milk. Washing – 3x 10 minutes in PBST (0.05%). Developed by Clarity Western ECL substrate (Biorad) and exposed to film. comment to the image: line 1 – K562 positive control line 2 – S2 cells control line 3 – S2 cells with silenced RUVBL2 RUVBL2 should show band around 50 kDa, however in S2 cells there wasn’t any band about this size even with high concentration of antibody and loading of 30 μg of protein sample. In K562 leukemic cells there was a band above 55 kDa (MW marker PageRuler Plus Prestained Protein Ladder, Thermo Scientific). There were strong bands around 30 kDa and also double band around 120 kDa (in S2 cells). |
Product Page for CHCHD3 antibody – middle region (ARP57040_P050)
Researcher: Jin-Mi Heo
Application: IP
Species+tissue/cell type: Lane 1: 20ug HEK2932T lysate + scRNA
Lane 2: 20ug HEK2932T lysate + siRNA1
Lane 3: 20ug HEK2932T lysate + siRNA2
Lane 4: 20ug HEK2932T lysate + siRNA3
Lane 5: 20ug HEK2932T lysate + siRNA4
Primary antibody dilution:1:1000
Secondary antibody: Anti-rabbit-HRP
Secondary antibody dilution: 1:2000
Product Page for TRAK1 antibody – middle region (ARP54520_P050)
Researcher: Jin-Mi Heo
Application: IP
Species+tissue/cell type: Lane 1: 400ug HEK2932T lysate
Lane 2: 400ug HEK2932T IP’d w/IgG control
Lane 3: 400ug HEK2932T IP’d w/TRAK1 ab
Primary antibody dilution:1:1000
Secondary antibody: Anti-rabbit-HRP
Secondary antibody dilution: 1:2000
Product Page for RHOT2 Antibody (ARP62300_P050)
Researcher: Jin-Mi Heo
Application: Western Blotting
Species+tissue/cell type:
Lane 1: 20ug untransfected HEK293T
Lane 2: 20ug RHOT2 transfected HEK293T
Primary antibody dilution:1:1000
Secondary antibody: Anti-rabbit-HRP
Secondary antibody dilution: 1:2000
