PGK1 Antibody [H17D18] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: Hela, Lane 2: NIH/3T3, Lane 3: C6, Lane 4: HuH7

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat

Source
Rabbit Monoclonal Antibody

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
43 kDa

Positive Control	HeLa cells; NIH/3T3 cells; C6 cells; HCC1806 cells; HuH-7 cells; LAMA-84 cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
PGK1 Antibody [H17D18] detects endogenous levels of total PGK1 protein.

Subcellular Location
Cytoplasm, Mitochondrion

Uniprot ID
P00558

Clone
H17D18

Synonym(s)
Phosphoglycerate kinase 1; Cell migration-inducing gene 10 protein; Primer recognition protein 2 (PRP 2); PGK1; PGKA

Background
PGK1 (phosphoglycerate kinase 1) is a core glycolytic enzyme that catalyzes the reversible transfer of a phosphate group from 1,3‑bisphosphoglycerate to ADP, generating ATP and 3‑phosphoglycerate and thereby linking the upper and lower halves of the glycolytic pathway in most mammalian cells. PGK1 also functions as a non‑canonical protein kinase that is itself regulated by post‑translational modifications, including autophosphorylation at Tyr324, which is required for its full activation and is reversed by the lipid and protein phosphatase PTEN; loss of PTEN in tumor cells consequently elevates phospho‑Tyr324‑PGK1, enhances glycolytic flux, and promotes tumor growth. In hypoxic or nutrient‑stressed conditions, PGK1 can translocate to the mitochondria where it phosphorylates the pyruvate dehydrogenase kinase PDK1 at Thr338, inhibiting the pyruvate dehydrogenase complex, suppressing mitochondrial pyruvate oxidation, and favoring lactate production and glycolytic dependency. Under glutamine‑starved or hypoxic stress, acetylated PGK1 interacts with Beclin1 and phosphorylates it to activate the VPS34 complex and initiate autophagy, thereby coordinating metabolic adaptation with selective degradation and recycling of cytoplasmic components. PGK1 expression and activity are upregulated in multiple cancer types, where its dual glycolytic and kinase functions support proliferation, invasion, and stress survival, and altered PTEN‑dependent regulation of PGK1 Tyr324‑phosphorylation is linked to aggressive brain‑tumor phenotypes and poor clinical outcomes.

Background

PGK1 (phosphoglycerate kinase 1) is a core glycolytic enzyme that catalyzes the reversible transfer of a phosphate group from 1,3‑bisphosphoglycerate to ADP, generating ATP and 3‑phosphoglycerate and thereby linking the upper and lower halves of the glycolytic pathway in most mammalian cells. PGK1 also functions as a non‑canonical protein kinase that is itself regulated by post‑translational modifications, including autophosphorylation at Tyr324, which is required for its full activation and is reversed by the lipid and protein phosphatase PTEN; loss of PTEN in tumor cells consequently elevates phospho‑Tyr324‑PGK1, enhances glycolytic flux, and promotes tumor growth. In hypoxic or nutrient‑stressed conditions, PGK1 can translocate to the mitochondria where it phosphorylates the pyruvate dehydrogenase kinase PDK1 at Thr338, inhibiting the pyruvate dehydrogenase complex, suppressing mitochondrial pyruvate oxidation, and favoring lactate production and glycolytic dependency. Under glutamine‑starved or hypoxic stress, acetylated PGK1 interacts with Beclin1 and phosphorylates it to activate the VPS34 complex and initiate autophagy, thereby coordinating metabolic adaptation with selective degradation and recycling of cytoplasmic components. PGK1 expression and activity are upregulated in multiple cancer types, where its dual glycolytic and kinase functions support proliferation, invasion, and stress survival, and altered PTEN‑dependent regulation of PGK1 Tyr324‑phosphorylation is linked to aggressive brain‑tumor phenotypes and poor clinical outcomes.

References
https://pubmed.ncbi.nlm.nih.gov/28486006/ https://pubmed.ncbi.nlm.nih.gov/31492635/

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%