Presentation Title

Optimization of Immunoprecipitation Protocol of Protein hnRNPH2 from Melanoma Cells

Speaker Credentials

OMS-II

Speaker Credentials

DO

College

Dr. Kiran C. Patel College of Osteopathic Medicine, DO

Location

Nova Southeastern University, Davie, Florida, USA

Format

Poster

Start Date

21-2-2020 8:30 AM

End Date

21-2-2020 4:00 PM

Abstract

Objective: Despite advances in melanoma drug discovery, the average survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets 1. Introduction: We identified heterogeneous nuclear ribonucleoprotein (hnRNP) H2 as such a target (Palrasu et al., Cell Physiol Biochem 2019;53:656-86). Mechanistic investigations showed that targeting hnRNPH2 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. Our project focused on optimization of isolating and purifying hnRNPH2 from melanoma cells. Methods: Antibody-bead crosslinking, immunoprecipitation (IP), silver stain, Coomassie stain, and western blots were used. Results: Initial experiments showed low levels of isolated H2. In order to obtain greater quantity of the protein, we scaled up antibody/bead complex 10 times resulting in higher H2 levels in eluate. Overall, we were able to isolate 10ug of pure H2. In the future, we are planning to obtain 100ug of pure H2 to perform protein biochemistry and biophysics. Conclusions: It was shown that multiple cycles of IP along with higher scales of antibody/bead complex ratio were required to obtain sufficient amount of the protein to be visible on the western blot stain. 1Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, 3321 College Avenue, CCR r.612, Fort Lauderdale, FL 33314 2 Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, 3321 College Avenue, Fort Lauderdale, FL 33314 3 Halmos College of Natural Sciences and Oceanography, 8000 N Ocean Dr, Dania Beach, FL 33004

4 Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, 3321 College Avenue, Fort Lauderdale, FL 33314 1. Foundation MR. Melanoma Facts and Stats 2018. Available from: https://www.melanoma.org/understand-melanoma/what-melanoma/melanoma-facts-and-stats.

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Feb 21st, 8:30 AM Feb 21st, 4:00 PM

Optimization of Immunoprecipitation Protocol of Protein hnRNPH2 from Melanoma Cells

Nova Southeastern University, Davie, Florida, USA

Objective: Despite advances in melanoma drug discovery, the average survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets 1. Introduction: We identified heterogeneous nuclear ribonucleoprotein (hnRNP) H2 as such a target (Palrasu et al., Cell Physiol Biochem 2019;53:656-86). Mechanistic investigations showed that targeting hnRNPH2 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. Our project focused on optimization of isolating and purifying hnRNPH2 from melanoma cells. Methods: Antibody-bead crosslinking, immunoprecipitation (IP), silver stain, Coomassie stain, and western blots were used. Results: Initial experiments showed low levels of isolated H2. In order to obtain greater quantity of the protein, we scaled up antibody/bead complex 10 times resulting in higher H2 levels in eluate. Overall, we were able to isolate 10ug of pure H2. In the future, we are planning to obtain 100ug of pure H2 to perform protein biochemistry and biophysics. Conclusions: It was shown that multiple cycles of IP along with higher scales of antibody/bead complex ratio were required to obtain sufficient amount of the protein to be visible on the western blot stain. 1Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, 3321 College Avenue, CCR r.612, Fort Lauderdale, FL 33314 2 Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, 3321 College Avenue, Fort Lauderdale, FL 33314 3 Halmos College of Natural Sciences and Oceanography, 8000 N Ocean Dr, Dania Beach, FL 33004

4 Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, 3321 College Avenue, Fort Lauderdale, FL 33314 1. Foundation MR. Melanoma Facts and Stats 2018. Available from: https://www.melanoma.org/understand-melanoma/what-melanoma/melanoma-facts-and-stats.