- PA5-63081 - Invitrogen Antibodies VGF antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: U2OS cells (WT and VGF KO) were treated with 0.3 μg/mL of Brefeldin A (Product # 00-4506-51) for 18 hrs to block secretion. Western blot of VGF was performed by loading 100 µg of WT and VGF KO non-treated or non-treated cell lysates in RIPA buffer onto a 4-15% 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. VGF was detected at approximately 68 kDa (designated by the black arrow) using a VGF polyclonal antibody (Product # PA5-63081) at a dilution of 1:500 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: U2OS cells (WT and VGF 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 VGF 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 concentrated using centrifugal filter 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 with a membrane NMWL of 50 kDa. Immunoblots were performed with 100 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). VGF was detected at approximately 68 kDa using a VGF polyclonal antibody (Product # PA5-63081) 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 the peroxidase-conjugated secondary antibody (Product # 65-6120) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of VGF in human cell line MCF7 shows positivity in vesicles. Samples were probed using a VGF Polyclonal Antibody (Product # PA5-63081).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of VGF in human cell line SK-MEL-30 using VGF Polyclonal Antibody (Product # PA5-63081) shows localization to the Golgi apparatus.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of VGF in human colon using VGF Polyclonal Antibody (Product # PA5-63081).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of VGF in human testis using VGF Polyclonal Antibody (Product # PA5-63081).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of VGF in human hypothalamus using VGF Polyclonal Antibody (Product # PA5-63081).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of VGF in human colon, hypothalamus, pituitary gland and testis using VGF Polyclonal Antibody (Product # PA5-63081) (A) shows similar protein distribution across tissues to an independent VGF Polyclonal Antibody (B).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of VGF in human liver using VGF Polyclonal Antibody (Product # PA5-63081) shows low expression as expected.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of VGF in human cerebral cortex using VGF Polyclonal Antibody (Product # PA5-63081) shows high expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of VGF in human pituitary gland using VGF Polyclonal Antibody (Product # PA5-63081).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: U2OS cells were washed 3x with PBS and starved for ~18 hrs in DMEM high glucose containing L-glutamate and penicillin/streptomycin. Immunoprecipitation of VGF was performed on U2OS concentrated cell culture media, concentrated by 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 concentrated using centrifugal filter units 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 with a membrane NMWL of 50 kDa. Antibody-bead conjugates were prepared by adding 1 µg of VGF polyclonal antibody (Product # PA5-63081) with 30 µL of protein A-Sepharose beads and rocked overnight at 4°C. 0.85 mg of lysate 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 VGF polyclonal antibody. 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.

PA5-63081