Glutamate Dehydrogenase 1/2 Antibody (Rabbit mAb) [D16D18]

Application: Reactivity: Mouse, Rat, Human

Lane 1: HepG2, Lane 2: Mouse brain, Lane 3: Mouse liver, Lane 4: Rat liver

Experiment Essentials

WB
Recommended WB dilution ratio: 1:10000

Usage Information

Dilution
WB1:1000 - 1:10000 IHC1:250 - 1:500

Application
WB, IHC

Reactivity
Mouse, Rat, Human

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
61 kDa

Positive Control	Human fetal liver tissue; Human normal tonsil tissue; Human normal brain tissue; Human normal kidney tissue; Human normal colon tissue; Human liver tissue; Human skeletal muscle tissue; HepG2 cells; HeLa cells; 293T 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes. 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) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 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) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.

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) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

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) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), 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.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
Glutamate Dehydrogenase 1/2 Antibody (Rabbit mAb) [D16D18] detects endogenous levels of total Glutamate Dehydrogenase 1 and Glutamate Dehydrogenase 2 protein.

Subcellular Location
Endoplasmic reticulum, Mitochondrion

Uniprot ID
P00367, P49448

Clone
D16D18

Synonym(s)
GLUD, GLUD1, GDH 1, GLUDP1, GLUD2, GDH 2, Glutamate Dehydrogenase 1, Glutamate Dehydrogenase 2

Background
Glutamate Dehydrogenase 1 (GLUD1) and Glutamate Dehydrogenase 2 (GLUD2) encode closely related mitochondrial glutamate dehydrogenases that catalyze the reversible oxidative deamination of glutamate to α‑ketoglutarate and ammonia, positioning these enzymes at a key junction between amino acid catabolism, the tricarboxylic acid cycle, and nitrogen handling in liver, brain, and endocrine tissues. The proteins share a conserved hexameric allosteric dehydrogenase architecture, with catalytic sites that bind glutamate and NAD(H)/NADP(H) and regulatory sites that respond to activators such as ADP and leucine and inhibitors such as GTP, allowing GLUD1/2 activity to be tightly tuned by cellular energy charge and amino acid availability and thereby to adjust anaplerotic flux of carbon into the TCA cycle and mitochondrial redox state. In pancreatic β cells, GLUD1 activity couples amino acid metabolism to insulin secretion: oxidative deamination of glutamate increases α‑ketoglutarate supply to the TCA cycle, enhances ATP production, and promotes closure of ATP‑sensitive K⁺ channels and depolarization, so gain‑of‑function GLUD1 mutations that impair GTP‑mediated inhibition cause the hyperinsulinism–hyperammonemia (HI/HA) syndrome, characterized by leucine‑sensitive hyperinsulinemic hypoglycemia and chronically elevated plasma ammonia due to increased renal ammoniagenesis. Patients with HI/HA carry dominant activating GLUD1 variants that increase enzyme responsiveness to leucine and reduce GTP inhibition, and they display protein‑triggered hypoglycemia, fasting hypoglycemia, and persistent mild hyperammonemia, while brain involvement with developmental delay and epilepsy in some cases is consistent with enhanced glutamate dehydrogenase activity also affecting neuronal glutamate handling and neurotransmission. Regulation of GLUD1 and GLUD2 in nerve tissue is complex, with transcriptional control, alternative promoters, and post‑translational modulation integrating signals from energy status, amino acid supply, and calcium, so that neuronal and glial GDH tune glutamate oxidation versus recycling in concert with glutamine synthetase and transaminases to maintain excitatory neurotransmitter pools and mitochondrial energy production. GLUD1 and GLUD2 function as allosterically regulated mitochondrial dehydrogenases that convert glutamate and NAD(P)⁺ into α‑ketoglutarate, reduced pyridine nucleotides, and ammonia, linking amino acid flux to TCA cycle anaplerosis, insulin secretion, nitrogen balance, and neuronal energy metabolism, and GLUD1 gain‑of‑function mutations in HI/HA syndrome illustrate how disruption of this regulatory system drives a defined endocrine–metabolic disease phenotype.

Background

Glutamate Dehydrogenase 1 (GLUD1) and Glutamate Dehydrogenase 2 (GLUD2) encode closely related mitochondrial glutamate dehydrogenases that catalyze the reversible oxidative deamination of glutamate to α‑ketoglutarate and ammonia, positioning these enzymes at a key junction between amino acid catabolism, the tricarboxylic acid cycle, and nitrogen handling in liver, brain, and endocrine tissues. The proteins share a conserved hexameric allosteric dehydrogenase architecture, with catalytic sites that bind glutamate and NAD(H)/NADP(H) and regulatory sites that respond to activators such as ADP and leucine and inhibitors such as GTP, allowing GLUD1/2 activity to be tightly tuned by cellular energy charge and amino acid availability and thereby to adjust anaplerotic flux of carbon into the TCA cycle and mitochondrial redox state. In pancreatic β cells, GLUD1 activity couples amino acid metabolism to insulin secretion: oxidative deamination of glutamate increases α‑ketoglutarate supply to the TCA cycle, enhances ATP production, and promotes closure of ATP‑sensitive K⁺ channels and depolarization, so gain‑of‑function GLUD1 mutations that impair GTP‑mediated inhibition cause the hyperinsulinism–hyperammonemia (HI/HA) syndrome, characterized by leucine‑sensitive hyperinsulinemic hypoglycemia and chronically elevated plasma ammonia due to increased renal ammoniagenesis. Patients with HI/HA carry dominant activating GLUD1 variants that increase enzyme responsiveness to leucine and reduce GTP inhibition, and they display protein‑triggered hypoglycemia, fasting hypoglycemia, and persistent mild hyperammonemia, while brain involvement with developmental delay and epilepsy in some cases is consistent with enhanced glutamate dehydrogenase activity also affecting neuronal glutamate handling and neurotransmission. Regulation of GLUD1 and GLUD2 in nerve tissue is complex, with transcriptional control, alternative promoters, and post‑translational modulation integrating signals from energy status, amino acid supply, and calcium, so that neuronal and glial GDH tune glutamate oxidation versus recycling in concert with glutamine synthetase and transaminases to maintain excitatory neurotransmitter pools and mitochondrial energy production. GLUD1 and GLUD2 function as allosterically regulated mitochondrial dehydrogenases that convert glutamate and NAD(P)⁺ into α‑ketoglutarate, reduced pyridine nucleotides, and ammonia, linking amino acid flux to TCA cycle anaplerosis, insulin secretion, nitrogen balance, and neuronal energy metabolism, and GLUD1 gain‑of‑function mutations in HI/HA syndrome illustrate how disruption of this regulatory system drives a defined endocrine–metabolic disease phenotype.

References
https://pubmed.ncbi.nlm.nih.gov/19690084/ https://pubmed.ncbi.nlm.nih.gov/22658952/

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%