The Bovine 2,3-Bisphosphoglycerate (2,3-BPG) ELISA Kit from Assay Genie is specifically designed for the accurate quantification of 2,3-BPG levels in bovine serum, plasma, and tissue homogenates. This kit offers superior sensitivity and specificity, ensuring precise and reliable results for a variety of research applications.2,3-BPG is a key intermediate in the glycolytic pathway and is essential for regulating oxygen release from hemoglobin in red blood cells. Abnormal levels of 2,3-BPG have been associated with various physiological and pathological conditions, including hypoxia, anemia, and metabolic disorders.
By accurately measuring 2,3-BPG levels, researchers can gain valuable insights into the metabolic status and oxygen transport capacity of bovine samples.Overall, the Bovine 2,3-BPG ELISA Kit provides researchers with a powerful tool for studying the role of 2,3-BPG in bovine physiology and pathology, ultimately advancing our understanding of oxygen homeostasis and metabolic regulation in bovine species.
2,3-Bisphosphoglyceric acid (2,3-Bisphosphoglycerate or 2,3-BPG, also known as 2,3-diphosphoglycerate or 2,3-DPG) is a three-carbon isomer of the glycolytic intermediate 1,3-bisphosphoglyceric acid (1,3-BPG). 2,3-BPG is present in human red blood cells (RBC; erythrocyte) at approximately 5 mmol/L. It binds with greater affinity to deoxygenated hemoglobin (e.g. when the red cell is near respiring tissue) than it does to oxygenated hemoglobin (e.g., in the lungs) due to spatial changes: 2,3-BPG (whose size is estimated at about 9 angstroms) fits in the deoxygenated hemoglobin configuration (11 angstroms), but not as well in the oxygenated (5 angstroms). It interacts with deoxygenated hemoglobin beta subunits by decreasing their affinity for oxygen, so it allosterically promotes the release of the remaining oxygen molecules bound to the hemoglobin, thus enhancing the ability of RBCs to release oxygen near tissues that need it most. 2,3-BPG is thus an allosteric effector.
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant general 2,3-Bisphosphoglycerate
Storage:
Please see kit components below for exact storage details
Note:
For research use only
Components
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
60µL
-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.
Step
1.
Add 50µL 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.
2.
Immediately add 50µL 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) and let it sit in the well for 1-2 minutes. 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 45 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 terminate the 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 amicro-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.
