4MID® ProAKAP4 ELISA Kits
ProAKAP4
Mature A-kinase anchor protein 4 (AKAP4) and its precursor proAKAP4 are specific structural and functional proteins of the spermatozoa. ProAKAP4 is full-length AKAP4 that is processed to form the mature AKAP4 protein after after removal of some amino acids. The AKAP4 gene belongs to the large family of A-kinase anchor proteins (AKAPs), which have unique cellular localization and ability to form complexes with other signaling molecules, thereby enabling precise and spatial signaling cascades to discrete subcellular compartments. Unlike other AKAPs, proAKAP4 and AKAP4 have a strict localization in the flagellum of sperm cells. The AKAP4 polypeptide is expressed in a variety of mammalian species and is highly conserved with overall 70% of homology.
ProAKAP4 and Male Reproduction
ProAKAP4 is an intracellular protein found only in spermatozoa and not in seminal plasma. Both proAKAP4 and AKAP4 are specifically localized to the fibrous sheath of the principal piece of the sperm flagellum. AKAP4 is one of the major components (about 50%) of the fibrous sheath and plays an important role in the assembly of the fibrous sheath, chemotaxis, capacitation and motility of sperm. ProAKAP4 is highly conserved among species, and its specific regulation during spermatogenesis seems to be crucial for the analysis of overall semen quality. Therefore, proAKAP4 is considered to be a functional marker of spermatozoa.
4MID® Kits for the Quantification of ProAKAP4
Amerigo Scientific offers ELISA kits for the specific detection and quantification of proAKAP4 in spermatozoa of different species. The 4MID® kit is a quantitative sandwich ELISA assay designed to detect and quantify the biomarker proAKAP4 in sperm samples. Assessment of semen functionality using the 4MID® kit can improve reproductive success in the setting of assisted reproductive technology. The 4MID® kit is composed of a 96-well plate coated with a monoclonal antibody specific for proAKAP4.
| Product Name |
Details |
| Pig 4MID® Kit (proAKAP4 marker)
|
Specificity: Pig
Sample type: Fresh, Chilled or Frozen Sperm |
| Horse 4MID® Kit (proAKAP4 marker)
|
Specificity: Horse / Donkey
Sample type: Fresh, chilled and frozen semen |
| Bull 4MID® Kit (proAKAP4 marker)
|
Specificity: Bull
Sample type: Fresh, chilled or frozen semen / Isolated spermatozoa |
| Dog 4MID® Kit (proAKAP4 marker)
|
Specificity: Dog
Sample type: Fresh, chilled and frozen spermatozoa / straw |
| Rabbit 4MID® Kit (proAKAP4 marker)
|
Specificity: Rabbit
Sample type: Fresh, chilled or frozen semen (straws) |
| Ram 4MID® Kit (proAKAP4 marker)
|
Specificity: Ram
Sample type: Fresh, chilled and frozen semen / isolated spermatozoa |
| Rodent and Human 4MID® Kit (proAKAP4 marker)
|
Specificity: Rodent / Human
Sample type: Fresh, frozen sperm / isolated spermatozoa |
| Goat 4MID® Kit (proAKAP4 marker)
|
Specificity: Goat
Sample type: Fresh, chilled and frozen semen / isolated spermatozoa |
| Cat 4MID® Kit (proAKAP4 marker)
|
Specificity: Cat
Sample type: Fresh, chilled and frozen semen / isolated spermatozoa |
| Camel 4MID® Kit (proAKAP4 marker)
|
Specificity: Camel
Sample type: Fresh, chilled and frozen semen / isolated spermatozoa |
| Mouse 4MID® Kit (proAKAP4 marker)
|
Specificity: Murine / All rodents
Sample type: Isolated spermatozoa/ fresh/ frozen / straws |
Amerigo Scientific also offers 4MID® kit in format BB (2 tests), which allows analysis of 12 to 24 semen per kit , with 1 or 2 tests per strip run.
Applications of 4MID® Kits
The concentration of proAKAP4 is an objective indicator of sperm quality, as well as sperm function and longevity. ProAKAP4 concentrations can be measured using the 4MID® kit to select semen before cryopreservation or to predict sperm motility after thawing. High proAKAP4 concentration in a semen sample indicates that the semen is of high quality, durable motility and good spermatogenesis.
Figure 1. 4MID kits for optimization of semen cryopreservation
Figure 2. ProAKAP4 concentration as a functional indicator of sperm
Operation Procedure of 4MID® Kits
Samples like fresh, frozen or dilute semen are lysed with the Spermatozoa Lysis Buffer and then pipetted into the wells. Then proAKAP4 binds to the antibody-coated plate and is detected by a Detection Antibody covalently coupled to horseradish peroxidase. A Substrate Solution is added to each well and color level appears proportionally to the amount of proAKAP4. The color reaction is stopped by the Stop Solution and the color intensity is measured by spectrophotometry at 450 nm. A Lyophilized Standard available in the 4MID® kit is used to perform a reference curve to determine the concentration of proAKAP4 present in each sperm sample.
Figure 3. Procedure for for determination of proAKAP4 concentrations in semen samples using the 4MID® ELISA kit
Characterization of the ProAKAP4 Expression
In a mouse testis, the results of immunostaining using a specific monoclonal antibody against proAKAP4 showed that proAKAP4 staining only occurred in round spermatids, elongated spermatids, and spermatozoa.
Figure 4. Characterization of the ProAKAP4 expression in a mouse testis
Shelf-life of Chilled Semen Evaluation Using Pig 4MID® Kits
ProAKAP4 concentrations in chilled semen of three pigs were assessed by Pig 4MID Kits. The semen from three boars were conditioned with the same extender and follow up during 7 days to assess proAKAP4 amount variations in time (shelf-life).
Figure 5. Shelf-life of chilled semen evaluation using pig 4MID® kits
ProAKAP4 conversion into AKAP4 was observed up day 7 for boars with high and normal proAKAP4 concentrations at Day 0 (Pig 1 and Pig 2). In contrast, proAKAP4 concentration was rapidly decreased at Day 1 in pig semen with initial low concentrations of proAKAP4 (Pig 3). ProAKAP4 concentrations may be regulated by factors such as nutriments, ROS expression, oxidative stress, etc. occurring in the environment of the living boar sperm cells and the initial cell machinery.
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