- MIA1609 - Invitrogen Antibodies APOB antibody

Askeland A, Borup A, Østergaard O, Olsen JV, Lund SM, Christiansen G, Kristensen SR, Heegaard NHH, Pedersen S
Biomedicines 2020 Jul 25;8(8)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Apolipoprotein B Monoclonal Antibody (F2C9)(Product # MIA1609) and 500 and 270kDa band corresponding to Apolipoprotein B was observed across sample tested. Whole cell extracts (40 µg lysate) of Human Plasma (Lane 1) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). 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 (2 µg/mL concentration) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 Apolipoprotein B was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Apolipoprotein B (F2C9) Mouse Monoclonal Antibody (Product # MIA1609) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Apolipoprotein B was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Apolipoprotein B Monoclonal Antibody (F2C9) (Product # MIA1609) at 4 µg/mL concentration in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (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 cytoplasm localization. Panel e represents negative cell line U-87 MG. 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: Figure 5 Difference in the co-isolation of contaminants such as serum albumin, lipoproteins, and other non-EV proteins between isolates produced by high-speed centrifugation (Cent. ), size-exclusion chromatography (SEC), and peptide affinity precipitation (PAP). ( A ) High-speed centrifugation produced EV isolates with the highest abundance of serum albumin. ( B ) SEC isolates contained the highest amount of lipoproteins. ( C ) Immunoblotting confirms elevated levels of apolipoprotein B in isolates produced by SEC. ( D ) PAP isolates contained the highest amount of non-EV proteins. Error bars: Mean +- SD. Significance levels: * ( p -value < 0.05) and ** ( p -value < 0.01).

MIA1609