- PA1-187 - Invitrogen Antibodies UBC antibody

Submitted references A nuclear-based quality control pathway for non-imported mitochondrial proteins.

Shakya VP, Barbeau WA, Xiao T, Knutson CS, Schuler MH, Hughes AL
eLife 2021 Mar 18;10

Uncoupling of p97 ATPase activity has a dominant negative effect on protein extraction.

Rycenga HB, Wolfe KB, Yeh ES, Long DT
Scientific reports 2019 Jul 17;9(1):10329

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration.

Lobanova ES, Finkelstein S, Li J, Travis AM, Hao Y, Klingeborn M, Skiba NP, Deshaies RJ, Arshavsky VY
Nature communications 2018 Apr 30;9(1):1738

p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair.

Fullbright G, Rycenga HB, Gruber JD, Long DT
Molecular and cellular biology 2016 Dec 1;36(23):2983-2994

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Ubiquitin was performed by loading 20 µg of the indicated whole cell lysates per well, and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. Ubiquitin was detected at ~8.5 kDa after probing with a Ubiquitin polyclonal antibody (Product # PA1-187) at a dilution of 1:1000 in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, washing in TBST, and probing with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). NOTE: In addition to detecting ubiquitin monomer, this antibody also detects poly-ubiquitinated proteins at various molecular weights.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Ubiquitin was performed on HeLa cells either left untreated (right panel) or treated with 10uM of MG132 proteasome inhibitor (left panel) for 2 hours at 37C. Whole cell lysates were prepared using IP Lysis buffer (Product # 87787), and 10 µg of the lysates along with 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) were loaded onto a Novex® 10-20% Tricine Gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288) and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. Ubiquitin was detected at ~8.5 kDa after probing with a Ubiquitin polyclonal antibody (Product # PA1-187) at a dilution of 1:1000 in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, washing in TBST, and probing with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). NOTE: In addition to detecting ubiquitin monomer, this antibody also detects poly-ubiquitinated proteins at various molecular weights.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. Nuclear protein quality control degrades non-imported mitochondrial proteins. ( A ) Western blot of yeast expressing Ilv2-GFP +- FCCP +- MG-132. ( B ) Western blot of yeast expressing Ilv2-GFP +- FCCP in wild-type (WT) and E3 KO strains. ( C ) Western blots showing cycloheximide (CHX) chase of Ilv2-GFP in WT and E3 KO strains in the presence of FCCP. ( D ) Western blot showing ubiquitylation of immunoprecipitated Ilv2-GFP +- FCCP in WT and E3 KO strains. Pgk1 = loading control. E3 KO = san1 Delta ubr1 Delta doa10 Delta. P = precursor and M = mature. Figure 3--figure supplement 1. Nuclear protein quality control promotes non-imported mitochondrial protein degradation. ( A ) Western blot of yeast expressing Ilv2-HA +- FCCP +- MG-132. ( B, C ) Western blots of yeast expressing the Ilv2-HA ( B ) or endogenous Ilv2 ( C ) +- FCCP in WT and E3 KO strains. ( D ) Western blots of yeast expressing Ilv2-GFP +- FCCP in WT and the indicated mutant yeast strains. ( E, F ) Western blots showing the CHX chase of Ilv2-HA ( E ) or endogenous Ilv2 ( F ) in WT and E3 KO strains in the presence of FCCP. ( G ) Western blots of yeast expressing the Lat1-GFP +- FCCP in WT and E3 KO strains. P = precursor form, M = mature form. Pgk1 = loading control. Figure 3--figure supplement 2. Nuclear protein quality control promotes non-imported mitochondrial protein degradation. ( A ) Yeast expressing the indicated GFP and mCherry tagged mitochondrial proteins +- FCCP. ( B ) Quantification of ( A ).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 p97-R155C is functionally defective for chromatin extraction. ( A ) pDNA was incubated in extract supplemented with p97-WT, p97-D1D2, or p97-R155C. DNA-bound proteins were isolated by plasmid pull-down at various times and visualized by Western blot with the indicated antibodies. Note that p97 antibodies recognize both endogenous and recombinant proteins. ( B , C ) Relative quantitation of total DNA-bound p97 ( B ) and ubiquitin ( C ) from three biological replicates. Values are normalized to peak accumulation in the +p97-WT reaction and error bars represent +/- one standard deviation. Full Western blot images are available in Fig. S7 .

PA1-187

Antibody data