Giantin (Golgi Marker) Antibody [F5N17]

Catalog No.: F1556

Application: Reactivity: Human

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

使用情報

Dilution
IF1:200-1:400

Application
IF

Source
Mouse Monoclonal Antibody

Reactivity
Human

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

ポジティブコントロール	HeLa cells; Hek293 cells
ネガティブコントロール

プロトコール

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

生物学的記述

Specificity
Giantin (Golgi Marker) Antibody [F5N17] detects endogenous levels of total Giantin protein.

タンパク質の局在
ゴルジ装置、細胞内膜系

Uniprot ID
Q14789

Clone
F5N17

Synonym(s)
Golgin subfamily B member 1; 372 kDa Golgi complex-associated protein (GCP372); Giantin; Macrogolgin; GOLGB1

Background
Giantin (GOLGB1), also known as Giantin Golgi protein, is the largest mammalian golgin, a family of long coiled-coil proteins localized primarily to the cis/medial Golgi and serving as a key structural marker of Golgi integrity. Giantin consists of an extended rod-like coiled-coil cytoplasmic domain that forms a rigid scaffold, a C-terminal transmembrane anchor, and interaction motifs for proteins such as p115, GM130, GRASP65, Rab1, and SNAREs, enabling it to tether vesicles without reliance on specific key residues. It regulates Golgi architecture by inhibiting excessive fusion and interconnections between stacks and cisternae, ensuring the formation of discrete reaction compartments for sequential glycosylation; knockdown of Giantin leads to reduced fenestrae size and number, accelerated membrane protein diffusion, and increased cisternal connectivity, which slows intra-Golgi transport and impacts protein and lipid glycosylation. Biologically, Giantin tethers COPI vesicles to the cis-Golgi, facilitates ER-Golgi and intra-Golgi trafficking through complexes with coat proteins, and supports Golgi reassembly after mitosis or disruption by agents like brefeldin A or ethanol. It coordinates the secretory pathway for surface and lysosomal protein sorting, with its loss altering glycosylation patterns and cargo kinetics. Disease associations include Congenital Disorders of Glycosylation (CDG), connective tissue disorders, ciliopathies (from dynein-2 mislocalization), and impaired secretion due to trafficking defects.

Background

Giantin (GOLGB1), also known as Giantin Golgi protein, is the largest mammalian golgin, a family of long coiled-coil proteins localized primarily to the cis/medial Golgi and serving as a key structural marker of Golgi integrity. Giantin consists of an extended rod-like coiled-coil cytoplasmic domain that forms a rigid scaffold, a C-terminal transmembrane anchor, and interaction motifs for proteins such as p115, GM130, GRASP65, Rab1, and SNAREs, enabling it to tether vesicles without reliance on specific key residues. It regulates Golgi architecture by inhibiting excessive fusion and interconnections between stacks and cisternae, ensuring the formation of discrete reaction compartments for sequential glycosylation; knockdown of Giantin leads to reduced fenestrae size and number, accelerated membrane protein diffusion, and increased cisternal connectivity, which slows intra-Golgi transport and impacts protein and lipid glycosylation. Biologically, Giantin tethers COPI vesicles to the cis-Golgi, facilitates ER-Golgi and intra-Golgi trafficking through complexes with coat proteins, and supports Golgi reassembly after mitosis or disruption by agents like brefeldin A or ethanol. It coordinates the secretory pathway for surface and lysosomal protein sorting, with its loss altering glycosylation patterns and cargo kinetics. Disease associations include Congenital Disorders of Glycosylation (CDG), connective tissue disorders, ciliopathies (from dynein-2 mislocalization), and impaired secretion due to trafficking defects.

References
[1] Satoh A, et al. Front Cell Dev Biol. 2019 Aug 27;7:160. [2] Linstedt AD, et al. Mol Biol Cell. 1993 Jul;4(7):679-93.

PVDFメンブレン孔径参考表

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%