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NSM 706: Research Methods I Dr.

Banerjee Biochemical and Molecular Techniques (Resubmitted Paper)

Umme Begum 05/ 15/ 2011

Western Blotting and its Application in Neuroscience


Western blotting is also referred to as protein blotting or immunoblotting and is an extension of the other blotting procedures, namely, Southern blotting which is used to separate DNA and Northern blotting which is used to identify RNA. In the western blotting procedure, the separated proteins are transferred from a sodium dodecyl sulfate polyacrylamide gel (SDSPAGE) to the absorbent membranes mentioned above. This technique is very useful in detecting and characterizing those proteins that are not found in abundance. The fact that, wet membranes are pliable and easy to handle, only a small amount of reagent is required for transfer analysis, multiple replicas of the gel is possible and the same protein transfer can be used for multiple successive analysis makes western blotting very advantageous (Kurien, ScoWeld, 2006). Proteins can be analyzed once the measure of its functional activity is known, when the protein level can be measured using its known antibody, or when the protein itself is not known but its regulatory protein it interacts with is known. Overall, western blotting is used when the molecular weight of the protein and the antibody that specifically binds to it is known. For protein analysis using the Western blotting technique, the functional activity of the proteins and their three dimensional structure and subunit association must be intact after homogenizations. Application of mild conditions for homogenization is required to keep the functional activity of the proteins. Western blotting requires three steps: sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfer of the proteins to nitrocellulose or nylon membrane and finally visualization of the protein bands. After the cells are homogenized, aliquots of the lysate are separated by size using SDS-PAGE. Once the proteins have been separated by SDS-PAGE, they are then transferred to a nitrocellulose or nylon membrane. This is achieved via placing the gel between two buffer soaked filter papers and applying an electrical current in the direction of transfer, namely, from the cathode to the anode. To visualize the proteins immunoblotting is necessary. Once the proteins have been transferred to the nitrocellulose of nylon membrane, the membrane is washed with blocking agent, this prevents non-specific binding of antibodies. The membrane is then probed for the protein of interest with a specific primary antibody. The membrane is then again probed a second time. The second probe is an antibody that is specific for the primary antibody and is then conjugated to a detectable enzyme: the site of the protein of interest is thus tagged with an enzyme that is specific to both the primary and secondary antibody. The product thus leave a colored trace at the site of the band or spot representing the protein of interest.
Cai, Q., Zhu, Z., Huang, S., Li, H., Fan, X., Jia, N., Zhang, B., Song, L., Li, Q., Liu, J. Sex and region difference of the expression of ERK in prenatal stress offspring hippocampus. Int. J. Devl. Neuroscience. 2007. 25, 207-213.

Introduction: It has been reported that prenatally stressed animals present with hippocampal damage, increasing anxiety, depressive behavior, delay in motor reflex development, learning impairments, increased basal corticosteriod concentration and impaired HPA axis. Other reports suggest that the prenatal restraint stress (PNS) might alleviate depressive behavior in females, but not in males. Prenatal stress is shown to cause neuronal loss and oxidative damage in the hippocampus of rat offspring. The hippocampus is involved in the regulation of the HPA axis and the behavioral responses associated with stress. It is evidenced that repeated restraint stress suppresses neurogenesis in the adult rat dentate gyrus; specifically PNS has been shown to inhibit cell proliferation in the dentate gyrus of the hippocampus and reduce hippocampal volume. It was seen that PNS causes an increase in the concentration of the intracellular calcium and the production of ROS in hippocampal CA3 region. One of the additional pathways of the MAPK pathway, the ERK pathway has been reported to be involved in the initiation of hippocampal long term potentiation. It has also been reported that oxidative stress activates MAPK cascades also, EKR stimulations by ROS has shown up in neurons. The following study investigates the effects of PNS on ROS (radioactive species) in hippocampal CA3 region of female offspring. The hypothesis of the following study is as follows: that PNS may affect the expression of MAPK in offspring hippocampi, which has shown to be the baseline for behavioral/cognitive affects. The aim of the following study involves determining the effects of prenatal restraint stress on the expression of p-ERK and total ERK in offspring

NSM 706: Research Methods I Dr. Banerjee Biochemical and Molecular Techniques (Resubmitted Paper)

Umme Begum 05/ 15/ 2011

hippocampus. Materials and Methods: 15 female and 5 male Sprague-Dawley rats were used. The pregnant rats of PNS group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 minutes. To prevent habituation, restraint periods were randomized. After postnatal day 30, the rats were decapitated under deep anesthesia with chloral hydrate (400mg/kg). The hippocampus was dissected and homogenized. The total protein concentration in each sample was determined using the BCA protein assay reagent kit. The sample proteins were then separated by electrophoresis by SDS-PAGE at room temperature. After electrophoresis, the proteins in the gel were transferred to 0.45mm polyvinyldiflouride membranes (PVDF). The membrane was blocked with 5% fat-free dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1hr at room temperature and then incubated with the primary antibody. ERK was analyzed by western blotting using specific antibodies against the active (biphosphorylated) forms of each enzyme. The total ERK levels were evaluated with rabbit polyclonal antibodies that recognize both unphosphorylated and phosphorylated ERK by probing with another PVDF membrane. Reactive proteins were detected with an ECL Western Blotting Detection Kit. Immunohistochemistry was performed for the hippocampus for qualitative evaluations of MAPK. The immunoreactions were performed by treating the sections of the hippocampi with DAB kit. Results and Discussion: The preceding study have identified that prenatal restraint stress leads to increased expression of p-ERK and EKK in female offspring hippocampi. The immunohistochemical analysis demonstrate that exposure of pregnant rats to stress during the last week of gestation results in an increased level of ERK in the hippocampal CA1, CA3 and CA4 regions of female offspring. The analysis also demonstrates that there is an increase in ERK in the hippocampal CA3 region of PNS male offspring. The increased expression of ERK seen under PNS implies that corticosterone might have a role in the regulation of ERK in the developing hippocampus. It is also suggested from the results that females are more prone to be affected by PNS than males. The developing brain is significantly impacted by endogenous or exogenous glucocorticoids during hippocampal development. PNS females had a higher overall adrenocorticotropic hormone response to restraint stress than to female controls. Findings suggest that an effect of PNS on the HPA axis is more predominant in females than in males. Reasons for CA3 being more vulnerable to PNS could be due to dentate gyrus expressing a high density of both Type 1 and Type II coriticosteroid receptors, these same neurons project to the CA3 pyramidal cells via the mossy fibers. It is reported that oxidative stress activates MAPK cascades, therefore EKR stimulation by ROS has been seen. Overall, PNS affected offspring hippocampal synaptic plasticity associated ERK/MAPK cascades.
Wu, Y., Wang, X-F., Mo, X., Sun, H., Li, J., Zen, Y., Lin, T., Yuan, J., Xi, Z., Zhu, X., Zheng, J. Expression of laminin 1 in hippocampi of patients with intractable epilepsy. Neuroscience Letters. 2008. 443, 160-164.

Introduction: Epilepsy is a disorder of recurrent seizures that often underlies some sort of neurological condition. While it is agreed among scientists that repeated seizures cause sprouting of axons, the link between the two is still under investigation. Increased axonal growth may cause excitatory abnormal neural networks. Laminin is one of the important elements of the extra cellular matrix and it regulates cell activity in the CNS. Laminins structure and role make it suitable to relay information in the developing embryo. During CNS development, axon sprouting establishes proper neuronal migration and neural connections. Studies have shown that over expression of laminin can promote axonal growth in cultured peripheral nerves. Laminin is expressed in the CNS only during development and is not expressed in adult CNS, however it was seen that patients with recurrent epilepsy have increased expression in laminin 1 mRNA in CNS of adult patients. This study was undertaken to evaluate whether laminin 1 participates in the axonal structural remodeling in patients with recurrent/intractable epilepsy. Laminin 1 mRNA and protein expression in hippocampal tissues of patients with intractable epilepsy patients was evaluated using fluorescence quantitative PCR (FQ-PCR), immunoflourescence, immunohistochemistry microscopy and western blotting.

NSM 706: Research Methods I Dr. Banerjee Biochemical and Molecular Techniques (Resubmitted Paper)

Umme Begum 05/ 15/ 2011

Methods and materials: Hippocampal tissue samples from 10 patients with intractable epilepsy (16 to 58 years old) were obtained. All of the patients had hippocampal sclerosis, characterized by the disappearance of hippocampal neurons and glial/fibrous hyperplasia. The types of seizure in the study included complex partial seizures and generalized tonic-clonic seizures. For comparison, six histologically normal hippocampus samples from cadavers that have no CNS abnormalities, no history of epilepsy or use of antiepileptic drugs were used. Experiments and control groups were analyzed through Western blotting and the ratio of the band intensities of laminin 1 and actin (laminin 1/ actin) from the same electrophoresis gel was analyzed. The procedures for the western blotting of the hippocampi were as follows: tissues were snap-frozen in liquid nitrogen, and protein was extracted directly by homogenization in SDS sample buffer containing 10% SDS and a mixture of protease inhibitors. The protein bands were electrotransferred to a polyvinylidene diflouride membrane. The membranes were divided into two parts to detect the laminin subunit blocked with 3% bovine serum albumin in PBS for 1h and then incubated for 2h at room temperature in PBS. To determine whether post mortem delay can lead to a reduction in laminin 1, a preliminary evaluation of the control tissues was analyzed by western blotting. Results and Discussion: As shown by the results, the hippocampal slices of the patients with intractable epilepsy, laminin 1 mRNA and protein expression were elevated compared to the normal. The results also indicate that laminin 1 may play a role in the development of drug resistance to epileptic seizures. Proposal for Future Research and application of the Western Blot Technique (add the last part): Autism is a neuropsychiatric disorder that affects 1 in 150 children. Currently there is an absence of consistent biomarkers for the diagnosis of autism. For this reason, diagnosis of autism is based on well characterized core behavioral symptoms. Among them, abnormal social interactions, social communication, repetitive behaviors and/or restrictive interests have been consistently reported. Current research in autism strives to identify any relevant diagnostic marker for autism. Animal models have been extremely useful in testing genetic hypothesis and in evaluating proposed treatments. For autism in particular, the preferred animal model used are transgenic mice. The inbred strain of mice of paramount interest are the BTBR T+tf/J (BTBR). BTBRs show significant behavioral similarities as have been reported in individuals with autism. They exhibit lower levels of play soliciting behaviors as juveniles and lack sociability in the adult social approach task. The BTBR also emit significantly fewer ultrasonic vocalizations in various social settings and also displays high levels of repetitive self-grooming. Perhaps the most under represented area of research involving the BTBR are their repetitive and obsessive compulsive behavior. They have been reported to display high numbers of self grooming and long durations of self grooming sessions that often exceed the usual 1 minute self grooming. Currently this unique autistic-relevant phenotype is being investigated. Fluoxetine and risperidone are among the many drugs that have been used to treat symptoms associated with autism. Risperidone is an atypical antipsychotic that blocks D2 and 5HT2A receptors is approved by the US Food and Drug Administration (FDA) to reduce repetitive behavior and self injurious behavior in children with autism (Chadman, 2010). Selective serotonin reuptake inhibitors (SSRIs) are considered the first line treatment for repetitive and obsessive compulsive behavior seen in OCD and in individuals with autism. SSRIs are a group of pharmacological agents that inhibit CNS neuronal uptake of serotonin through the reuptake of 5HT and increasing 5HT levels in the synapse by blocking reuptake of 5HT by the presynaptice nerve terminal. Of the many proposed etiology of autism, serotonin dysregulation is one of the more plausible theories for the core symptoms that are associated with autism. The core symptoms of repetitive and stereotyped pattern of behavior have been understudied in comparison to other core symptoms of autism. The study that I am focusing on involves the use of different levels of Risperidone administered to both BTBR (experimental group) and C57/BL/6J (control group). The aim of the study is to determine if Risperidone has any affect on the OCD behavior in Marble Burying seen in the BTBR mice models.

NSM 706: Research Methods I Dr. Banerjee Biochemical and Molecular Techniques (Resubmitted Paper)

Umme Begum 05/ 15/ 2011

References: Abcam Laboratories : www.abcam.com/technical Cai, Q., Zhu, Z., Huang, S., Li, H., Fan, X., Jia, N., Zhang, B., Song, L., Li, Q., Liu, J. Sex and region difference of the expression of ERK in prenatal stress offspring hippocampus. Int. J. Devl. Neuroscience. 2007. 25, 207-213. Kurien, T. B., Scofield, H. R. Western Blotting. Methods. 2006. 38, 283-293. Wu, Y., Wang, X-F., Mo, X., Sun, H., Li, J., Zen, Y., Lin, T., Yuan, J., Xi, Z., Zhu, X., Zheng, J. Expression of laminin 1 in hippocampi of patients with intractable epilepsy. Neuroscience Letters. 2008. 443, 160-164.

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