Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [4]
- Immunohistochemistry [2]
- Other assay [1]
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Validation data
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- Product number
- MA5-32538 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Midkine Recombinant Rabbit Monoclonal Antibody (JF096-5)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Description
- Recombinant rabbit monoclonal antibodies are produced using in vitro expression systems. The expression systems are developed by cloning in the specific antibody DNA sequences from immunoreactive rabbits. Then, individual clones are screened to select the best candidates for production. The advantages of using recombinant rabbit monoclonal antibodies include: better specificity and sensitivity, lot-to-lot consistency, animal origin-free formulations, and broader immunoreactivity to diverse targets due to larger rabbit immune repertoire.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- JF096-5
- Vial size
- 100 µL
- Concentration
- 1 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of Midkine was achieved by transfecting PANC-1 with Midkine specific siRNAs (Silencer® select Product # S8626, S8627). Western blot analysis was performed using conditioned media from knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The conditioned media was quantified using Pierce™ Rapid Gold BCA Protein Assay Kit (Product # A53225) and normalized prior to loading. The blot was probed with Midkine Recombinant Rabbit Monoclonal Antibody (JF096-5) (Product # MA5-32538, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Midkine.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Midkine Recombinant Rabbit Monoclonal Antibody (JF096-5) (Product # MA5-32538) and an 18 kDa band corresponding to Midkine was observed in conditioned medium of PANC-1 and SH-SY5Y cells but not in conditioned medium of HL-60 and RT4. 7 µl of conditioned media of PANC-1 (Lane 1), SH-SY5Y (Lane 2), HL-60 (Lane 3) and RT-4 (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- HAP1 cells were treated with 0.3 µg/mL of Brefeldin A (Product # 00-4506-51) for 18 hrs. Western blot of MDK was performed by loading 50 µg of HAP1 (WT and MDK KO) treated and non-treated cell lysates in RIPA buffer onto a large 10-20% gradient polyacrylamide gel. Proteins on the blots were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. MDK was detected (designated by the black arrow) using an MDK recombinant monoclonal antibody (Product # MA5-32538) at a dilution of 1:1,000 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4°C. The peroxidase-conjugated secondary antibody (Product # 65-6120) was diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr at room temperature. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106). Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- HAP1 cells (WT and MDK KO) were washed 3x with PBS and starved for ~18 hrs in DMEM high glucose containing L-glutamate and penicillin/streptomycin. Western blot analysis of Midkine was performed by collecting culture media and centrifuging for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were initially concentrated using centrifugal filter units by centrifuging at 4000 x g for 15 min. The resulting 500 µL of the concentrated media were centrifuged again at 4000 x g for 15 min using centrifugal filter units to 200 µL. Immunoblots were performed with ~30 µg of proteins from concentrated culture media on large 10-20% gradient polyacrylamide gels. Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. Proteins on the blots were visualized with Ponceau staining (below immunoblot). MDK was detected at approximately 15 kDa using a MDK recombinant monoclonal antibody (Product # MA5-32538) at a dilution of 1:500 in 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST) overnight at 4°C followed by secondary antibody diluted to 0.2 µg/mL using Goat anti-Rabbit IgG (H+L) HRP antibody (Product # 62-6520). Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106). Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemical analysis of Midkine of paraffin-embedded Human liver cancer tissue using a Midkine Monoclonal antibody (Product #MA5-32538). Counter stained with hematoxylin.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemical analysis of Midkine of paraffin-embedded Human pancreas tissue using a Midkine Monoclonal antibody (Product #MA5-32538). Counter stained with hematoxylin.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- HAP1 cells (WT and MDK KO) were washed 3x with PBS and starved for ~18 hrs in DMEM high glucose containing L-glutamate and penicillin/streptomycin. Immunoprecipitation of Midkine was performed by collecting culture media and centrifuging for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were initially concentrated using centrifugal filter units by centrifuging at 4000 x g for 15 min. The resulting 500 µL of the concentrated media were centrifuged again at 4000 x g for 15 min using centrifugal filter units to 200 µL. Antibody-bead conjugates were prepared by adding 1 µg of MDK recombinant monoclonal antibody (Product # MA5-32538) with 30 µL of protein G-Sepharose beads and rocked overnight at 4°C. 0.3 mg of concentrated culture media was incubated with an antibody-bead conjugate for 2 hours at 4°C. Following centrifugation and multiple washes, 10% starting material (SM), 10% unbound fraction (UB) and immunoprecipitated fraction (IP) were processed for immunoblot using the same MDK recombinant monoclonal antibody at 1:200. Ponceau stained transfer of blot is shown. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.