LF-MA0118

antibody from Invitrogen Antibodies

Targeting: UBC

Western blot (Supportive data in Antibodypedia)

ELISA (Recommended by provider)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Recommended by provider)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

LF-MA0118

Provider

Invitrogen Antibodies

Product name

Anti-Ubiquitin Monoclonal Antibody (2A1)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

A suggested positive control for this product is Bosc23 cells transfected with GFP-Ub.

Reactivity

Human

Host

Mouse

Isotype

IgG

Antibody clone number

2A1

Vial size

100 µL

Concentration

1.0 mg/mL

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole-cell extracts (30 µg lysate) of MCF7 (Lane 1), and MCF7 treated with MG132 (20 µM for 1 hour) (Lane 2), Jurkat (Lane 3) and Jurkat treated with MG132 (20 µM for 1 hour) (Lane 4). The blots were probed with Mouse Anti-Ubiquitin B Monoclonal Antibody (Product # LF-MA0118, 0.5 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A ladder-like pattern corresponding to ubiquitinated proteins were observed across the cell lines tested. Upon treatment with MG132, there was an increase in the detection of ubiquitinated proteins. This can be attributed to the MG132-induced inhibition of proteasomal machinery, which under normal conditions degrades the ubiquitin-tagged proteins. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Ubiquitin B was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Ubiquitin (2A1) Mouse Monoclonal Antibody at 1:250 dilution 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) at 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic and nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of Ubiquitin B was done on SH-SY5Y cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Ubiquitin B Mouse Monoclonal Antibody (Product # LF-MA0118, red histogram) or with mouse isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Rabbit Anti-Mouse Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.