This document discusses the purification of regucalcin (RGN), a calcium-binding protein, from seminal vesicular fluid. It introduces RGN and its functions in maintaining calcium homeostasis and roles in apoptosis and antioxidant activity. The authors describe using PCR, Western blot, and SDS-PAGE techniques to demonstrate that RGN is present in seminal fluid and confirm its identity using mass spectrometry. Their results show RGN can be purified from seminal fluid using an immunoaffinity method and identify its molecular weight. The conclusion states RGN's important roles and potential applications for developing cancer treatments.

1. Purification of Regucalcin from the
Seminal Vesicular Fluid A Calcium
Binding Multi-Functional Protein
By:
Ángela María Callejas
Ana Isabel Valencia Gómez
MOLECULAR BIOLOGY.

2. INTRODUCTION

3. INTRODUCTION
Seminal fluid
→ Grayish white bodily fluid.
→ Contains sperms which can fertilize the female ova, approximately 40-600 million
→ Volume of semen in an ejaculation: 2-5 ml.
The semen comprises of:
• Fructose
• Ascorbic acid
• Zinc
• Cholesterol
• Protein
• Calcium
• Chlorine
• Blood group antigens
• Citric acid
• Magnesium
• Vitamin B12
• Phosphorus
• Sodium
• Potassium
• Uric acid
• Lactic acid
• Nitrogen
• DNA

4. CALCIUM.
→ Ion.
→ 99% in the bone as crystals, 1% in LEC.
→ Nerve transmission> Liberation NT, and hormonal secretion
→ Neuromuscular junction> Acetyl hill.
→ Hemostasis and coagulation.
Required For vascular
contraction
and
vasodilation
INTRODUCTION

5. REGUCALCINA. (RGN)
Calcium binding protein.
Anti-apoptotic,anti-
prolific and anti-oxidative
functions.
Maintenance of intracellular
calcium homeostasis.
-Can mediate cell death
and proliferation by
modulating DNA synthesis
and fragmentation.
-Involved in the synthesis of
L-ascorbic acid that
scavenges the free
radicals.
INTRODUCTION

6. -Inhibition of cell divsion
and apoptotosis
suggesting
its role in maintaining
prostate cancer.
Is mainly secreted from
Sertoli cells: creates the
ideal conditions for
spermatogonial
development
Androgen target gene in
rat testis, play a role in
spermatogenesis

7. REGUCLACIN is in:
Glandular epitelium of the bulbouterine
and prostate gland
She half the
homeostasis of
CALCIUM
SEMINIFEROUS
TUBULAR FLUID
produced by: Sertoli
cells
MALE REPRODUCTIVE TRACT
RELATIONSHIP

8. GENERAL OBJECTIVE.
The objective of the present study is to develop an
immuno- affinity methodology to purify RGN from
seminal vesicular fluid.

9. MATERIALES Y
METODOS

10. PCR
→ Objetivo : Amplificación directa de un gen o fragmento de DNA o indirecta de un RNA
→ La técnica se basa en la amplificación in vitro, incremento geométrico del numero de copias
de una secuencia particular de DNA.
• Se deben aplicar los siguientes conceptos:
1. Desnaturalización de DNA
2. Hibridación de la hebra sencilla con un primer
3. Replicación de la hebra sencilla por una DNA polimerasa a partir del primer.
variaciones del método:
RT-PCR
RAPDs
PCR in situ
PCR-RFLP
SSCP
Entre otros.
MATERIALES Y METODOS

11. MATERIALES Y METODOS
DNA RECOMBINANTE.
→ El DNA se puede romper y unir con
fragmentos de la misma especie o de
una diferente.
Es necesario cortar (endonucleasa) y
unir (ligasas) el DNA.
• Las endonucleasas (enzimas de
restriccion): rompen la doble cadena
de DNA al reconocer una secuencia
especifica de bases. El rompimiento
es especifico y reproducible.
• Las ligasas unen dos cadenas de
DNA punta con punta.

12. MATERIALES Y MÉTODOS
Western blot o
inmunoblot:
→ Proteínas son separadas por
electroforesis, por medio de la técnica
SDS-PAGE poliacrilamida gel
electroforesis.
→ Luego se transfieren a un filtro
→ Incubar con anticuerpos
→ El anticuerpo unido al filtro se detecta
de varias maneras, identificando la
proteína contra la cual esta dirigido
el anticuerpo.

13. Cromatografía SDS-PAGE.
Electroforesis en gel de poliacrilamida con dodecilsulfato
sódico.
SDS desnaturaliza las proteínas,
eliminando sus estructuras secundaria y
terciaria.
Fraccionamiento que
obedece a: diferencia
de peso; longitud de la
cadena, y la forma de
la proteína.
Corriente eléctrica y
migración
Gel de
poliacrilamida
Tinción: Coomassie
blue o tinción
argéntea

14. Reacción antígeno-anticuerpo

15. RESULTADOS
PCR
WESTERN
BLOT
SDS-PAGE
SDS-PAGE
917 Pb
1.10h post
Inducción
2. Células no
inducidas
1. Purificada

16. RESULTADOS
SDS- PAGE WESTERN BLOT
1. No escindida (51KDa)
2. Escindida (33KDa)
1. rRGN, con AntiRGN

17. WESTERN BLOT
POLICLONAL
MONOCLONAL

18. RESULTADOS
WESTERN BLOT SDS- PAGE
1. Antes de
tratamiento
2. Tratado
3. Comprimidos
28KDa/ 34KDa/ 51KDa
Sobrenadante+
Anti-RGN

19. DISCUSSION
AUTHOR SAID YES/NO
-Xie Z, Schendel
S, Matsuyama
S, Reed JC
-Wang CK,
Lebowitz J,
Cheung HC
- Laurentino S,
Gonc¸alves J,
Cavaco JE,
Oliveira PF,
Alves MG, de
Sousa M,
Barros A,
Socorro S
• The elutes in SDS–PAGE showed a single band at 68
kDa in Coomassie R-250 staining. The acidic pH, used for
elution of the protein from the IAC has been speculated to
facilitate a conformational change in the protein favoring
the dimerisation.
• This is the first report of the presence of RGN in the
seminal vesicular fluid. Similar isoforms of RGN are
reported in rat liver tissue.

20. AUTHOR SAID YES/NO
-Parrish JJ,
Susko-Parrish
JLGJ
-Arun P, Aleti V,
Parikh K,
Manne V,
Chilukuri N
• Further, this protein had been confirmed as RGN by Liquid
Chromatography Mass Spectrometry/Mass Spectrometry
and has an average molecular weight of 33,878.07 Da
suggesting that the 68 kDa protein is a dimeric form of
RGN.
• A 917 bp PCR product had been amplified, including
primer flanking sequence of 36 bp and actual coding 881
bp DNA sequence of buffalo RGN. The amplified buffalo
RGN gene contained an open reading frame of 294 amino
acids, which is 5 amino acids truncated from N-terminal-
portion of the native RGN. The formation of low molecular
weight isoforms of RGN having 28 and 24 kDa by
proteolytic cleavage at the N-terminal end is reported.
DISCUSSION

21. CONCLUSION
→Proteins are responsible for the proper functioning
of the human body. In this article the importance of
RGN was exhibited, because it has a very important
role in the homeostasis of calcium, control of
apoptosis and anti-oxidative effect.
→This protein may help to control prostate cancer for
their actions in inhibition of cell cycle and apoptosis.

22. →The authors Demonstrated that RGN is in vesicle
seminal fluid by the utilization of PCR, western blot
and SDS PAGE and the confirmation with liquid
chromatography mass spectrometry.
→Being the regucalcin in the seminal fluid and have
important functions could be used in a future
synthetically for realization of drugs for the
treatment of cancer especially the male
reproductive tract.
CONCLUSION

25. • http://www.drelist.com/composition-of-human-semen/
• http://www.news-medical.net/health/What-is-Semen.aspx
• http://www.ncbi.nlm.nih.gov/pubmed/6151055
• http://lymediseaseguide.org/es/test-accuracy-western-blot
• Martínez Sánchez , Lina María; Vargas Grisales, Natalia;
Cardona Mejía, Laura; Osorio Ospina, Felipe; Ramírez
Pulgarin , Sergio. (29 de Julio de 2015). Biología Molecular.
En Biología molecular (octava edición) (pág. 160-201).
Medellín: Editorial Universidad Pontificia