Micro Methods Exam 1

what is the major function of tight junctions

maintain cellular polarity and act as a transmembrane barrier through selective permeability, only allowing specific ions and molecules into and out the cell,

what are the 3 major types of proteins found in tight junctions

JAMs, claudins, occludins

what are the major functions of claudins

act as the backbone of tight junctions

what is the structure of claudins

4 transmembrane helices, two extracellular loops (ECL 1 and 2) ECL 1 is larger, and both N- and C- terminals of the proteins are located within the cell membrane

functions of JAMs

cell barrier function, cell adhesion, cell movement

function of occludins

regulation of cell permeability

what were the major objectives in the pan et al paper?

characterize the expression and cellular distribution of claudin-3, 4 as normal endometrial cells progressed into endometriod acarcinoma and to determine correlation between expression of claudin-3, 4 and features of endometriod acarcinoma

steps of immunohistochemistry

1. fixation of tissues in formalin, embedded in paraffin, and sliced into thin sections

2. blocking and retrieval of antibodies through the use of buffers, heat, and blocking agents

3. staining with primary antibody to locate desired protein and then with secondary antibody to amplify signal

4. use of a substrate or counter staining to visualize antibodies

what was the experimental design of Dr Todds experiment

1. Isolate protein from normal and endometrial cancer cells 

2. Subject 30 micrograms of each protein to SDS-PAGE (X2 gels) 

3. Transfer proteins to two PVDF membranes

4. Incubate one membrane with an antibody to claudin-3 and one membrane with an antibody to claudin-4 (to detect the levels of these two proteins). 

5. After probing for claudin-3 and -4, incubate both membranes with an antibody to GAPDH (loading control)

what were the variables of Dr Todds experiement

Control: Normal endometrial cell lines

Experimental group: 9 late-stage endometrial cancer cell line groups

Loading control: GAPDH, to ensure the samples were loaded correctly

what is the difference between tissues and cell lines in cancer research

tissues are intact organs, body parts or tumors containing cancerous cells while cell lines are isolated cultured cancer cells

steps of immunoblotting

1. Isolate and extract proteins of interest from cells using buffers

2. Treatment of proteins with SDS-PAGE buffer to give proteins a negative net charge

3. Run proteins through gel electrophoresis to separate based on size

4. transfer proteins from gel to membrane

5. Treat proteins with a primary antibody that recognizes target protein

6. treat with a secondary antibody that targets the primary antibody

7. Add substrate that reacts with enzyme in secondary antibody to make fluorescent

what are the major components of a introduction section

background information about your topic that goes from broad to specific, evidence that your topic is relevant from other scientific papers, methods you plan to use to study this topic, main aim of your study

how do you cite papers in text

last name of each author and year published. If more than two authors use et al. or in place of author name use numbers in order of when you cited it that correspond to reference section

what are the major components of a reference section

author(s), title of source, date of publication, journal name, page number, issue number, date accessed

how do you cite papers in a reference section

list by author last name alphabetically or in numerical order as they were cited

what are the major components of a figure legend

A descriptive title, objective of the experiment, a brief materials and methods section, relevant results, and the any definitions or key to understanding figures

what are the major components of a discussion section

Interpretation of results, comparison to previous studies, implications, limitations, and future research directions

What is the prupose for a title in a scientific paper

convey the main findings of a research work and give readers an idea of the study