Antibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [4]
- Immunocytochemistry [1]
- Immunohistochemistry [2]
- Flow cytometry [3]
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- Product number
- PA5-78925 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Calpastatin Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- Reconstitute with 0.2 mL of distilled water to yield a concentration of 500 µg/mL.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µg
- Concentration
- 500 µg/mL
- Storage
- -20°C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot analysis of Calpastatin in Lane 1: COLO320 whole cell lysate using 50 µg (reducing conditions) per well. Electrophoresis was performed on 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours and protein was transferred to a nitrocellulose membrane at 150mA for 50-90 minutes. Sample was blocked with 5% Non-fat Milk/TBS for 1.5 hours at room temperature, incubated with Calpastatin polyclonal antibody (Product # PA5-78925) at a dilution of 0.5 µg/mL (overnight at 4°C), followed by goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10,000. Signal development was performed using a chemiluminescence (ECL) kit.
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot analysis of Calpastatin in, Lane 1: human Hela whole cell lysates, Lane 2: human Hacat whole cell lysates. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 µg of sample under reducing conditions. After Electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. The membrane was blocked with 5% non-fat milk/TBS for 1. 5 hour at RT. The membrane was incubated with Calpastatin Polyclonal Antibody (Product # PA5-78925) 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-rabbit IgG-HRP secondary antibody at a dilution of 1:5,000 for 1. 5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit. A specific band was detected for Calpastatin at approximately 120 kDa. The expected band size for Calpastatin is at 77 kDa.
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Knockdown of Calpastatin was achieved by transfecting HeLa cells with Calpastatin specific siRNAs (Silencer® select Product # s2398, s2399). Western blot analysis (Fig. a) was performed using whole cell extracts from Calpastatin knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Calpastatin Polyclonal Antibody (Product # PA5-78925, 0.5µg/ml) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Calpastatin.
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot was performed using Anti-Calpastatin Polyclonal Antibody (Product # PA5-78925) and bands at 80-130 kDa corresponding to Calpastatin was observed in all the cell lines tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1) and HeLa (Lane 2) were electrophoresed using Novex® NuPAGE® 4-12% % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Immunocytochemistry analysis of Calpastatin using anti-Calpastatin antibody (Product # PA5-78925) . Calpastatin was detected in a section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum and then incubated with 2μg/mL rabbit anti-Calpastatin antibody (Product # PA5-78925) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Immunohistochemistry analysis of Calpastatin on paraffin-embedded human intestinal cancer tissue. Antigen retrieval was performed using citrate buffer (pH6, epitope retrieval solution) for 20 mins. Sample was blocked using 10% goat serum, incubated with Calpastatin polyclonal antibody (Product# PA5-78925) with a dilution of 1 µg/mL (overnight at 4°C), and followed by biotinylated goat anti-rabbit IgG (30 minutes at 37°C). Development was performed using Streptavidin-Biotin-Complex (SABC) with DAB chromogen method.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of Calpastatin on paraffin-embedded human placenta tissue. Antigen retrieval was performed using citrate buffer (pH6, epitope retrieval solution) for 20 mins. Sample was blocked using 10% goat serum, incubated with Calpastatin polyclonal antibody (Product# PA5-78925) with a dilution of 1 µg/mL (overnight at 4°C), and followed by biotinylated goat anti-rabbit IgG (30 minutes at 37°C). Development was performed using Streptavidin-Biotin-Complex (SABC) with DAB chromogen method.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow Cytometry of Calpastatin in U2OS cells (blue line), isotype control rabbit IgG (green line) and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with Calpastatin Polyclonal Antibody (Product # PA5-78925) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow Cytometry of Calpastatin in A549 cells (blue line), isotype control rabbit IgG (green line) and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with Calpastatin Polyclonal Antibody (Product # PA5-78925) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow Cytometry of Calpastatin in A431 cells (blue line), isotype control rabbit IgG (green line) and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with Calpastatin Polyclonal Antibody (Product # PA5-78925) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.