Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [4]
- Immunocytochemistry [1]
- Immunohistochemistry [2]
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- Product number
- PA5-79347 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Hemoglobin alpha Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant full-length protein
- 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)
- Main image
- Experimental details
- Western blot analysis of Hemoglobin in Lane 1: rat spleen tissue lysate, Lane 2: U2OS 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 Hemoglobin polyclonal antibody (Product # PA5-79347) 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)
- Main image
- Experimental details
- Western blot analysis of Hemoglobin in Lane 1: rat spleen tissue lysate, Lane 2: U2OS 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 Hemoglobin polyclonal antibody (Product # PA5-79347) 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)
- Main image
- Experimental details
- Western blot analysis of Hemoglobin in, Lane 1: rat spleen tissue lysates, Lane 2: U2OS 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 50 µg of sample under reducing conditions. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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 Hemoglobin alpha Polyclonal Antibody (Product # PA5-79347) 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 1:10000 for 1. 5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit. A specific band was detected for Hemoglobin at approximately 15KD. The expected band size for Hemoglobin is at 15KD.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-Hemoglobin alpha Polyclonal Antibody (Product # PA5-79347) and a 15 kDa band corresponding to Hemoglobin alpha was observed only in K-562, HEL 92.1.7, Mouse Spleen and Mouse Bone Marrow which are reported to be positive and not in any other cell lines and tissues. Whole cell extracts (30 µg lysate) of K-562 (Lane 1), HEL 92.1.7 (Lane 2), HL-60 (Lane 3), Raji (Lane 4), U-2 OS (Lane 5) and A549 (Lane 6) and tissue extracts of Mouse Spleen (Lane 7), Mouse Bone Marrow (Lane 8) and Mouse Mammary gland (Lane 9) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). 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 (0.2 ug/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)
- Main image
- Experimental details
- Immunofluorescence analysis of Hemoglobin alpha was performed using 70% confluent log phase K-562 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Hemoglobin alpha Polyclonal Antibody (Product # PA5-79347) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Cytoplasmic localization. Panel e represents HL-60 cells having no expression of Hemoglobin. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemistry analysis of Hemoglobin on paraffin-embedded human liver 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 Hemoglobin polyclonal antibody (Product# PA5-79347) 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 Hemoglobin on paraffin-embedded human renal 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 Hemoglobin polyclonal antibody (Product# PA5-79347) 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.