Ubiquilin 2 Antibody (6H9) - BSA Free

Novus Biologicals | Catalog # NBP2-25164

Novus Biologicals
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Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Validated:

Human, Mouse, Rat

Cited:

Human, Mouse

Applications

Validated:

Knockout Validated, Immunohistochemistry, Immunohistochemistry-Frozen, Western Blot, Immunocytochemistry/ Immunofluorescence, Simple Western, Immunoprecipitation, Knockdown Validated

Cited:

Immunohistochemistry, Immunohistochemistry-Frozen, Western Blot, Immunocytochemistry/ Immunofluorescence, IF/IHC

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 Clone # 6H9

Format

BSA Free
View all Ubiquilin 2 Primary Antibodies »

Product Specifications

Immunogen

Human Ubiquilin 2 expressed in and purified from E. coli [UniProt# Q9UHD9]

Localization

Cytoplasm. Nucleus.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1

Theoretical MW

66-68 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Ubiquilin 2 Antibody (6H9) - BSA Free

Knockdown Validated: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Ubiquilin-2-Antibody-6H9-Knockdown-Validated-NBP2-25164-img0010.jpg
Simple Western: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Simple Western: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Simple Western: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - Simple Western lane view shows a specific band for Ubiquilin 2 in 0.5 mg/ml of HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Western Blot: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Western Blot: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Western Blot: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - Analysis of different tissue and cell lysates using mouse mAb to ubiquilin 2, NBP2-25164, dilution 1:1,000 in green: [1] protein standard (red), [2] NIH-3T3, [3] C6, [4] HEK293, [5] HeLa, [6] SH-SY5Y, [7] rat whole brain, and [8] mouse whole brain. The band at 65-70kDa corresponds to ubiquilin 2 protein, which is known to differ between the human and rodent proteins.
Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - Analysis of an NIH-3T3 cell culture stained with mouse mAb to ubiquilin 2, NBP2-25164, dilution 1:1,000 in green, and costained with chicken pAb to lamin A/C, dilution 1:5,000 in red. The blue is DAPI staining of nuclear DNA. The cells were treated with 50uM of chloroquine, an inhibitor of autophagy, for 16 hours prior to staining. NBP2-25164 antibody reveals vesicular staining of ubiquilin 2 protein accumulated in lysosomes in the cytoplasm, while the lamin A/C antibody stains the nuclear lamina.
Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - Analysis of Human U2OS cells stained with Ubiquilin 2 antibody. Primary antibody dilution: 1:1000. Image from verified customer review.
Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - HeLa cell cultures stained with NBP2-25164 (green) and chicken polyclonal antibody to Vimentin: NB300-223 (red). In most individual cells Ubiquilin 2 is present diffusely in the cytoplasm of cells, though some cells show enrichment of the protein in spherical autophagosome-like structure.
Knockout Validated: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Western Blot: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Western Blot: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - Lysates of HAP1 WT and Ubiquilin 2 KO were prepared, and 30 ug of protein were processed for immunoblot with NBP2-25164. The Ponceau stained transfer of the blot is shown. Antibody dilution used: 1/2000. Predicted band size: 65 kDa. Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).
Immunoprecipitation: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunoprecipitation: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunoprecipitation: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - HAP1 lysates were prepared, and immunoprecipitation was performed using 2.0 ug of Ubiquilin 2 antibody (NBP2-25164) pre-coupled to Dynabeads protein G. Samples were washed and processed for immunoblot with NBP2-25164. For immunoblot, Recombinant Ubiquilin 2 (Rb x Ubiquilin 2) was used at 1/1000. The Ponceau stained transfer of the blot is shown. SM=4% starting material; UB=4% unbound fraction. Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).
Knockout Validated: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164]

Immunocytochemistry/ Immunofluorescence: Ubiquilin 2 Antibody (6H9) [NBP2-25164] - HAP1 WT and Ubiquilin 2 KO cells were labeled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with glass bottom. Cells were stained with Ubiquilin-2 antibody (NBP2-25164) and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative image of the blue and red (grayscale) channels is shown. WT and KO cells are outlined with green and magenta dashed line, respectively. Antibody dilution used: 1/1000. Bars = 10 um Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).
Ubiquilin 2 Antibody (6H9) - BSA Free

Knockdown Validated: Ubiquilin 2 Antibody (6H9) - BSA Free [NBP2-25164] -

Heat Shock Generates Insoluble Ubiquitin-Positive Aggregates and Does Not Inactive Proteasomes, Related to Figure 1(A) UBQLN2 is not pelleted when cells are heat shocked post lysis. Cell lysates were incubated at 37 or 42C and then fractionated into soluble (S) and pellet (P) fraction. This indicates that UBQLN2 itself does not aggregate as a result of high temperature.(B) UBQLN2 levels are not upregulated in response to heat shock. HSP70 and GAPDH were used as a positive and negative controls, respectively.(C) Heat shock aggregates are insoluble in up to and including 1% SDS but are solubilized in 2% SDS. Blotting of soluble and pellet fractions with anti-ubiquitin and UQBLN2 antibodies confirmed dissolution of the aggregates in 2% SDS.(D) Proteasomes are active after heat shock. To confirm that proteasome activity was not affected by heat shock, we incubated U2OS and MEFs at the indicted temperatures for 2h. Cells were then harvested and cell lysates were incubated with the proteasome inhibitor MG132 or DMSO, followed by incubation with a fluorescent proteasome-activity probe, as indicated. The presence of fluorescently labeled beta-subunits at the same intensity under both heat stress and normal temperature, indicate that proteasome activity is not significantly affected by heat shock.(E) U2OS cells were treated with control or UBQLN2 siRNA and subjected to heat shock for the indicated times. Analysis of the pellet fraction revealed that insoluble ubiquitylated aggregates are generated within 5 min of heat shock, but that depletion of UBQLN2 does not noticeably alter the accumulation of these aggregates at any of the indicated time points. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27477512), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Ubiquilin 2 Antibody (6H9) - BSA Free

Knockdown Validated: Ubiquilin 2 Antibody (6H9) - BSA Free [NBP2-25164] -

Puromycin Does Not Upregulate UBQLN2 Levels and UBQLN2 Clears Nuclear Aggregated GFP-u, Related to Figure 2(A) Schematic representation on how the three fractions, total insoluble, nuclear soluble and total insoluble were generated.(B) Cells treated with puromycin were fractionated as indicated and treatment did not induce the nuclear localization of UBQLN2.(C) Puromycin treatment did not induce the upregulation of UBQLN2 protein.(D) Schematic showing the GFPu-NLS construct (Bennett et al., 2005).(E) HEK293 cells stably expressing GFPu-NLS were subject to heat shock for 2h at 42C and fractionated into soluble and insoluble fractions. GFPu-NLS recruited to the insoluble fraction after heat shock indicating its heat-induced aggregation.(F) GFPu-NLS cells were subject to heat shock for 2h at 42C and proteasome inhibition with 25 μM MG132 as indicated. UBQLN2 was immunoprecipitated and GFPu-NLS was found to co-immunoprecipitate only upon heat shock, consistent with UBQLN2 nuclear localization. Combined heat shock and proteasome inhibition increased the binding further.(G) GFPu-NLS cells were depleted of UBQLN2 or treated with a control non-targeting siRNA then treated with 50 μg/ml cycloheximide (CHX) for the indicated time to measure turnover. Turnover was quantified using data from three independent experiments.Error bars represent SE. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27477512), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for Ubiquilin 2 Antibody (6H9) - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:1000

Immunohistochemistry

1:1000

Simple Western

1:100

Western Blot

1:1000-1:2000
Application Notes
This Ubiquilin 2 (6H9). antibody is useful for Immunocytochemistry/Immunofluorescence, Immunohistochemistry, and Western Blot, where a band can be seen at approx. 66-68 kDa.

In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in HeLa lysate 0.5 mg/mL, separated by Size, antibody dilution of 1:100, apparent MW was 74 kDa. Use in Immunohistochemistry-Frozen reported in scientific literature (PMID:33028421).

Reviewed Applications

Read 2 reviews rated 5 using NBP2-25164 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

50% PBS, 50% glycerol

Format

BSA Free

Preservative

5mM Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at -20C. Avoid freeze-thaw cycles.

Background: Ubiquilin 2

Ubiquilin 2, also known as PLIC2 and Chap1, is a member of the ubiquilin protein family, which regulate the degradation of cellular proteins through proteasome or autophage-like pathways. Humans have four ubiquilin genes, each encoding a separate protein referred to as Ubiquilin 1, 2, 3 and 4. All ubiquilins contain an N-terminal ubiquitin-like (UBL) domain and a C-terminal ubiquitin-associated (UBA) domain, while the central part of the molecules are highly variable. The UBL domains bind subunits of the proteasome, and the UBA domains binds to polyubiquitin chains that are typically conjugated onto proteins marked for proteosomal degradation. Ubiquilin 2 has a unique region close to the C terminus containing 12 PXX tandem collagen like repeats, where P is proline and X is most cases valine, glycine, isoleucine or threonine. Teepu Siddique and his collaborators have identified mutations in the ubiquilin 2 gene leading to protein point mutations which were important contributors to several forms of amyotrophic lateral sclerosis (ALS) and Frontotemporal lobar degeneration (FTLD). Interestingly, these mutations involved alterations in proline residues in the PXX repeat region (P497H, P497S, P506T, P509S and P525S). Recently, the Lee and Trojanowski group investigated C9orf72 hexanucleotide expansion and ubiquilin 2 pathology in patients with ALS and FTLD by genetic analysis and immunohistochemistry and found distinct Ubiquilin 2 pathology in ALS and FTLD-TDP with C9orf72 expansion. C9orf72 hexonucleotide expansion is the most common cause to date of familial ALS and FTLD. Ubiquilin 2 protein is of different molecular size in mouse and human, 638 and 624 amino acids respectively. As a result the mouse protein, endogenously expressed in rodent 3T3 cells, runs on SDS-PAGE and western blots slightly slower than the human protein.

Alternate Names

Chap1, CHAP1/DSK2, DSK2, DSK2 homolog, FLJ10167, FLJ56541, hPLIC-2, N4BP4LIC-2, Nedd4 binding protein 4, PLIC-2, PLIC2Dsk2, Protein linking IAP with cytoskeleton 2, RIHFB2157, ubiquilin 2, ubiquilin-2, Ubiquitin-like product Chap1/Dsk2

Entrez Gene IDs

29978 (Human)

Gene Symbol

UBQLN2

OMIM

300264 (Human)

UniProt

Q9UHD9 (Human)

Additional Ubiquilin 2 Products

Product Documents for Ubiquilin 2 Antibody (6H9) - BSA Free

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Product Specific Notices for Ubiquilin 2 Antibody (6H9) - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for Ubiquilin 2 Antibody (6H9) - BSA Free

Customer Reviews for Ubiquilin 2 Antibody (6H9) - BSA Free (2)

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  • Ubiquilin 2 Antibody (6H9)
    Name: Anonymous
    Application: Immunoprecipitation
    Sample Tested: 293 whole cell lysate
    Species: Human
    Verified Customer | Posted 08/19/2022
    Lane 1,2: HEK293 whole cell lysate input (10 micrograms) Lane 3: HEK293 whole cell lysate immunoprecipitated with normal mouse IgG Lane 4: HEK293 whole cell lysate immunoprecipitated with Mouse anti-Ubiquilin-2 (6H9).
    Antibody was used at 1:30 dilution and was crosslinked to Protein A/G beads. HEK293 cells were lysed in 1x Tris buffered saline containing 1% NP40 and 5% glycerol. 600 micrograms of whole cell lysate was used for IP. IP was done for 2 hours.
    Ubiquilin 2 Antibody (6H9) - BSA Free NBP2-25164
  • Ubiquilin 2 Antibody (6H9)
    Name: Anonymous
    Application: Immunocytochemistry
    Sample Tested: U2OS cells
    Species: Human
    Verified Customer | Posted 02/09/2022
    Human U2OS cells
    Dilution: 1:1000
    Ubiquilin 2 Antibody (6H9) - BSA Free NBP2-25164

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