PA1-061

antibody from Invitrogen Antibodies

Targeting: COPB1 COPB

Provider product page for PA1-061

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [68]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [3]
Flow cytometry [1]
Other assay [14]

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Product number: PA1-061 - Provider product page
Provider: Invitrogen Antibodies
Product name: beta COP Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-061 detects Golgi beta-coatomer protein (beta-COP) from mouse, human and rat tissues.
Concentration: 4.4 mg/mL

COPB1 protein structure - PA1-061 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg) of Mouse Brain (Lane 1), NIH/3T3 (Lane 2), PC-3 (Lane 3), C2C12 (Lane 4), HeLa (Lane 5), HCT 116 (Lane 6) and COLO 205 (Lane 7). The blots were probed with Anti-beta COP Rabbit Polyclonal Antibody (Product # PA1-061, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~107 kDa band corresponding beta COP was observed across tissue and cell lines tested. 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 by Pierce™ Power Blotter System (22834). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of beta COP was achieved by transfecting HeLa with beta COP specific siRNAs (Silencer® select Product # S3372). Western blot analysis (Fig. a) was performed using Membrane Enriched extracts from the beta COP knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with beta COP Polyclonal Antibody (Product # PA1-061, 2ug/ml dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to beta COP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 1 The levels and subcellular distribution of GOLPH3 are different in different human breast cell lines. (A) Cell homogenates ( H ) from the indicated cell lines were used to prepare cytosolic ( C ) and membrane ( M ) fractions. Equivalent amounts of each fraction (10 mug of proteins) were subjected to SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (B) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in the cell homogenates as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in cytosolic ( C ) and membrane ( M ) fractions as shown in (A). Bar represents the mean +- standard deviation of the amount of immunoblot signal normalized with the signal for beta-actin, and also for the total amount of protein in each fraction (for more details see S1 Table ). *** P < 0.001; ns , not statistically significant.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 3 Cytosolic GOLPH3 behaves as a monomer in MCF 10A, MCF7, and MDA-MB-231 cells. Samples of a cytosolic fraction from the indicated cell lines were analyzed by gel filtration on a Superose 6 column. Arrows indicate the elution position of molecular size markers. Stokes radii ( R s ) are indicated in Angstroms ( A ). As a reference, the molecular mass ( M ) of molecular size markers is also indicated. Fractions were subjected to SDS-PAGE and immunobloting with antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 GalT-mCherry puncta localize to lysosomes ( a ) Hela cells expressing GalT-oxBFP (upper panels) or GalT-mCherry (lower panels) were fixed and stained with the GC marker anti-GM130. Colocalization was observed for GalT-oxBFP, but cells expressing moderate levels of GalT-mCherry exhibit both GC colocalization, and bright puncta throughout the cell. ( b ) GalT-mCherry expressing HeLa cells fixed and stained for the GC and intermediate compartment marker beta COP. The red puncta do not colocalize with the green beta COP puncta. ( c and d ) HeLa cells co-expressing GalT-moxGFP and GalT-mCherry show colocalization in the GC structure, but GalT-moxGFP does not localizes to red puncta. ( d ) Pretreatment with NH 4 Cl for 3 h leads to substantial colocalization of GalT-moxGFP with red puncta. ( e ) GalT-mcherry expressing HeLa cells were fixed and stained for the lysosomal marker anti-LAMP1. Several red puncta colocalize with the LAMP1 positive structures. Scale bars = 10 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 RAB29 rescues the G2019S LRRK2-mediated EGF trafficking deficits. ( A ) Top: Example of HeLa cell transfected with GFP-tagged RAB29 using Lipofectamine 2000 (LF2000), and stained with Golgi marker (beta-COP) and DAPI. Bottom: Example of HeLa cell co-transfected with GFP-tagged RAB29 and flag-tagged G2019S LRRK2 using LF2000, and stained with flag antibody and DAPI. Scale bar, 10 mum. ( B ) Cells were transfected with either pCMV, or cotransfected with flag-tagged G2019S LRRK2 and GFP or GFP-tagged RAB29 constructs as indicated, and surface-bound fluorescent EGF quantified. N = 3 independent experiments. * p < 0.05. ( C ) Cells were transfected with either pCMV, or cotransfected with flag-tagged G2019S LRRK2 and GFP or GFP-tagged RAB29 constructs, and internalized fluorescent EGF was quantified at 10 min (left) and 30 min (right) upon internalization. N = 3 independent experiments. * p < 0.05; ** p < 0.01. ( D ) HeLa cells were transfected with the indicated GFP-tagged RAB29 constructs using Lipofectamine 2000 (LF2000), and surface-bound fluorescent EGF quantified. N = 3 independent experiments. ( E ) Cells were transfected with the indicated GFP-tagged RAB29 constructs, and internalized fluorescent EGF was quantified at 10 min (left) and 30 min (right) upon internalization. N = 3 independent experiments. ( F ) HeLa cells were transfected with empty pCMV vector, with GFP-tagged RAB29 constructs, or with flag-G2019S LRRK2 along with GFP-tagged RAB29 constructs as indic

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 The G2019S LRRK2-mediated deficit in EGF trafficking is rescued by RAB29 independent of Golgi integrity. ( A ) Example of HeLa cell transfected with GFP-RAB29 using Lipofectamine 2000 (LF2000), and stained for beta-COP and DAPI in either the absence or presence of BFA treatment (5 mug/mL, 2 h) as indicated. Scale bar, 10 mum. ( B ) Example of HeLa cell cotransfected with GFP-RAB29 and flag-tagged G2019S LRRK2 using LF2000, and stained for flag and DAPI in either the presence or absence of BFA treatment (5 mug/mL, 2 h) as indicated. Scale bar, 10 mum. ( C ) HeLa cells were either transfected with pCMV (ctrl), flag-tagged G2019S LRRK2, GFP-tagged RAB29, or cotransfected with flag-tagged G2019S LRRK2 and GFP-tagged RAB29 using LF2000 as indicated, and either treated with or without BFA (5 mug/mL, 2 h) before determination of the amount of surface-bound fluorescent EGF. N = 3 independent experiments; * p < 0.05. ( D ) HeLa cells were either left untreated (ctrl), transfected with flag-tagged G2019S LRRK2, GFP-tagged RAB29, or cotransfected with flag-tagged G2019S LRRK2 and GFP-tagged RAB29 using LF2000, either treated with or without BFA (5 mug/mL, 2 h) as indicated, and internalized fluorescent EGF was quantified at 10 min (left) and 30 min (right) upon internalization. N = 3 independent experiments. * p < 0.05.