PLK1 mAb

Catalog No.: F3264

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使用情報

Dilution
WB1:1000 IF1:100 FCM1:50

Application
WB, IF, FCM

Source
Mouse

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

Predicted MW Observed MW
68 kDa 66 kDa
^*なぜ予測分子量と実際の分子量が異なるのか？下記の原因により、実際の分子量が予測と異なる：タンパク質の翻訳後修飾（リン酸化/糖鎖付加），スプライシングバリアント，イソフォーム，相対的な電荷，ポリマー。

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
PLK1 mAb recognizes endogenous levels of total PLK1 protein.

Synonym(s)
Plk,PLK1

Clone
F22N13

Background
Polo-like kinase 1 (Plk1) is a highly conserved serine/threonine kinase that plays a central role in regulating various stages of the cell cycle, particularly during mitosis. Structurally, Plk1 consists of an N-terminal catalytic kinase domain and a C-terminal polo-box domain (PBD), facilitating substrate recognition and localization to specific mitotic structures. Plk1 is essential for centrosome maturation, spindle assembly, chromosome alignment, sister chromatid segregation, and cytokinesis. It activates key mitotic regulators like Cdc25C and Myt1 through phosphorylation, ensuring proper cell division. Plk1 also participates in the DNA damage response by modulating checkpoint proteins and promoting recovery from the G2 DNA damage checkpoint. Its activity is regulated via phosphorylation, protein interactions, and subcellular localization, with peak activity occurring during the G2/M transition. Plk1 contributes to non-mitotic functions such as epithelial-to-mesenchymal transition (EMT), autophagy, apoptosis, and metabolic reprogramming. Plk1 phosphorylates glucose-6-phosphate dehydrogenase (G6PD), enhancing the pentose phosphate pathway to support biosynthesis in cancer cells. Dysregulation or overexpression of Plk1 is frequently observed in cancers and is associated with poor prognosis due to its role in promoting cell proliferation, transformation, EMT, and genomic instability. It also interacts with tumor suppressor p53 in an autoregulatory loop, inhibiting p53's pro-apoptotic functions while being transcriptionally repressed by it under DNA damage conditions.

Background

Polo-like kinase 1 (Plk1) is a highly conserved serine/threonine kinase that plays a central role in regulating various stages of the cell cycle, particularly during mitosis. Structurally, Plk1 consists of an N-terminal catalytic kinase domain and a C-terminal polo-box domain (PBD), facilitating substrate recognition and localization to specific mitotic structures. Plk1 is essential for centrosome maturation, spindle assembly, chromosome alignment, sister chromatid segregation, and cytokinesis. It activates key mitotic regulators like Cdc25C and Myt1 through phosphorylation, ensuring proper cell division. Plk1 also participates in the DNA damage response by modulating checkpoint proteins and promoting recovery from the G2 DNA damage checkpoint. Its activity is regulated via phosphorylation, protein interactions, and subcellular localization, with peak activity occurring during the G2/M transition. Plk1 contributes to non-mitotic functions such as epithelial-to-mesenchymal transition (EMT), autophagy, apoptosis, and metabolic reprogramming. Plk1 phosphorylates glucose-6-phosphate dehydrogenase (G6PD), enhancing the pentose phosphate pathway to support biosynthesis in cancer cells. Dysregulation or overexpression of Plk1 is frequently observed in cancers and is associated with poor prognosis due to its role in promoting cell proliferation, transformation, EMT, and genomic instability. It also interacts with tumor suppressor p53 in an autoregulatory loop, inhibiting p53's pro-apoptotic functions while being transcriptionally repressed by it under DNA damage conditions.

References
[1] Lee SY, et al. Dev Reprod. 2014 Mar;18(1):65-71.

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%