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Human Delta-aminolevulinic acid dehydratase (ALAD) ELISA Kit (HUEB2159)

SKU:
HUEB2159
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P13716
Range:
1.56-100 ng/mL
ELISA Type:
Sandwich
Synonyms:
ALAD, ALADH, Porphobilinogen synthase
Reactivity:
Human
$779
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Description

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Human Delta-aminolevulinic acid dehydratase (ALAD) ELISA Kit

The Human Delta-Aminolevulinic Acid Dehydratase (ALAD) ELISA Kit is a powerful tool for the precise measurement of ALAD levels in human biological samples, including serum, plasma, and cell culture supernatants. With exceptional sensitivity and specificity, this kit delivers accurate and consistent results, making it an indispensable resource for a variety of research endeavors.ALAD is a critical enzyme involved in heme biosynthesis, playing a crucial role in maintaining proper levels of heme in the body. Dysregulation of ALAD has been linked to various diseases, including lead poisoning and certain types of anemia.

By accurately measuring ALAD levels, researchers can gain valuable insights into these conditions and potentially discover new therapeutic targets.Whether investigating the impact of environmental toxins on ALAD activity or exploring the role of heme metabolism in disease pathology, the Human ALAD ELISA Kit offers a reliable and efficient solution for your research needs. Trust in its precision and performance to drive new discoveries and advancements in the field of biomedicine.

Product Name:Human Delta-aminolevulinic acid dehydratase (ALAD) ELISA Kit
SKU:HUEB2159
Size:96T
Target:Human Delta-aminolevulinic acid dehydratase (ALAD)
Synonyms:Porphobilinogen synthase, ALADH
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Human
Detection Range:1.56-100ng/mL
Sensitivity:0.78ng/mL
Intra CV:5.3%
Inter CV:10.0%
Linearity:
Sample1:21:41:81:16
Serum(N=5)82-92%99-108%108-120%103-113%
EDTA Plasma(N=5)104-113%113-123%100-112%109-120%
Heparin Plasma(N=5)101-111%88-98%110-120%101-113%
Recovery:
Sample TypeAverage(%)Recovery Range(%)
Serum10195-107
Plasma10397-109
Function:Catalyzes an early step in the biosynthesis of tetrapyrroles. Binds two molecules of 5-aminolevulinate per subunit, each at a distinct site, and catalyzes their condensation to form porphobilinogen.
Uniprot:P13716
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant human Delta-aminolevulinic acid dehydratase
Sub Unit:Homooctamer; active form. Homohexamer; low activity form.
Research Area:Metabolism
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:ALAD: Catalyzes an early step in the biosynthesis of tetrapyrroles. Binds two molecules of 5-aminolevulinate per subunit, each at a distinct site, and catalyzes their condensation to form porphobilinogen. Defects in ALAD are the cause of acute hepatic porphyria (AHEPP). A form of porphyria. Porphyrias are inherited defects in the biosynthesis of heme, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. They are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in red blood cells or in the liver. AHP is characterized by attacks of gastrointestinal disturbances, abdominal colic, paralysis, and peripheral neuropathy. Most attacks are precipitated by drugs, alcohol, caloric deprivation, infections, or endocrine factors. Belongs to the ALADH family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cofactor and Vitamin Metabolism - porphyrin and chlorophyll; EC 4.2.1.24; Lyase

Chromosomal Location of Human Ortholog: 9q32

Cellular Component: cytosol; extracellular region; nucleus

Molecular Function:catalytic activity; identical protein binding; porphobilinogen synthase activity; zinc ion binding

Biological Process: heme biosynthetic process; neutrophil degranulation; protein homooligomerization

Disease: Porphyria, Acute Hepatic

NCBI Summary:The ALAD enzyme is composed of 8 identical subunits and catalyzes the condensation of 2 molecules of delta-aminolevulinate to form porphobilinogen (a precursor of heme, cytochromes and other hemoproteins). ALAD catalyzes the second step in the porphyrin and heme biosynthetic pathway; zinc is essential for enzymatic activity. ALAD enzymatic activity is inhibited by lead and a defect in the ALAD structural gene can cause increased sensitivity to lead poisoning and acute hepatic porphyria. Alternative splicing of this gene results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Dec 2015]
UniProt Code:P13716
NCBI GenInfo Identifier:122833
NCBI Gene ID:210
NCBI Accession:P13716.1
UniProt Secondary Accession:P13716,Q16870, Q16871, Q9BVQ9, A8K375, B2R6F2,
UniProt Related Accession:P13716
Molecular Weight:36kDa
NCBI Full Name:Delta-aminolevulinic acid dehydratase
NCBI Synonym Full Names:aminolevulinate dehydratase
NCBI Official Symbol:ALAD
NCBI Official Synonym Symbols:PBGS; ALADH
NCBI Protein Information:delta-aminolevulinic acid dehydratase
UniProt Protein Name:Delta-aminolevulinic acid dehydratase
UniProt Synonym Protein Names:Porphobilinogen synthase
Protein Family:Aladin
UniProt Gene Name:ALAD
Component Quantity (96 Assays) Storage
ELISA Microplate (Dismountable) 8×12 strips -20°C
Lyophilized Standard 2 -20°C
Sample Diluent 20ml -20°C
Assay Diluent A 10mL -20°C
Assay Diluent B 10mL -20°C
Detection Reagent A 120µL -20°C
Detection Reagent B 120µL -20°C
Wash Buffer 30mL 4°C
Substrate 10mL 4°C
Stop Solution 10mL 4°C
Plate Sealer 5 -

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1. Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2. Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5. Repeat the wash process for five times as conducted in step 3.
6. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7. Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9. After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol
Serum If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
ELISA
Human ALAD ELISA Kit

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