Western blot (WB): | 1:500-2000 |
Immunohistochemistry (IHC): | 1:50-400 |
Immunofluorescence (IF): | 1:50-400 |
(Boiling the paraffin sections in 10mM citrate buffer,pH6.0,or PH8.0 EDTA repair liquid for 20 mins is required for the staining of formalin/paraffin sections.) Optimal working dilutions must be determined by end user. |
Figure 1. IHC analysis of Collagen IV using anti-Collagen IV antibody (M01411). Collagen IV was detected in paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Collagen IV Antibody (M01411) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.
Figure 2. IHC analysis of Collagen IV using anti-Collagen IV antibody (M01411). Collagen IV was detected in paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Collagen IV Antibody (M01411) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.
Figure 3. Western blot analysis of Collagen IV using anti-Collagen IV antibody (M01411). Electrophoresis was performed on a 8% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human HEK293T whole cell lysate,
Lane 2: human Hela whole cell lysate,
Lane 3: human A549 whole cell lysate. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1. 5 hour at RT. The membrane was incubated with mouse anti-Collagen IV antigen affinity purified monoclonal antibody (Catalog # M01411) at 0. 5 μg/mL overnight at 4°C, then washed with TBS-0. 1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1. 5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for Collagen IV at approximately 220KD. The expected band size for Collagen IV is at 161KD.
Figure 1. IHC analysis of Collagen IV using anti-Collagen IV antibody (M01411). Collagen IV was detected in paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Collagen IV Antibody (M01411) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.
Figure 2. IHC analysis of Collagen IV using anti-Collagen IV antibody (M01411). Collagen IV was detected in paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-Collagen IV Antibody (M01411) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.
Figure 3. Western blot analysis of Collagen IV using anti-Collagen IV antibody (M01411). Electrophoresis was performed on a 8% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human HEK293T whole cell lysate,
Lane 2: human Hela whole cell lysate,
Lane 3: human A549 whole cell lysate. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1. 5 hour at RT. The membrane was incubated with mouse anti-Collagen IV antigen affinity purified monoclonal antibody (Catalog # M01411) at 0. 5 μg/mL overnight at 4°C, then washed with TBS-0. 1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1. 5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for Collagen IV at approximately 220KD. The expected band size for Collagen IV is at 161KD.