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Sea Urchin Fertilization and MAPK

What happens when PLC is activated? Causes an influx of calcium ions from the ER to the cytoplasm
Calcium is a direct trigger for? exocytosis of cortical granules.
Cortical Granules contain? proteases that cut the protein tethering the vitelline envelope to the egg and muco-polysaccharide which produces an osmotic gradient, causing water to rush into this space. They also contain peroxidase which uses H2O2 to harden the envelope.
calcium ionophore Causes an artificial influx of Ca ions which induces exocytosis of the c.g.'s and other fert. events and produces activation w/out fertilization.
MAPK stands for Mitrogen Activated Protein Kinase
MAPK keeps oocytes in an arrested state until fertilization.
MAPK is turned on/off by.. phosphorylation/de-phosphorylation
What AA's must MAPK be phosphorylated at? Thr 202 and Tyr 204
Ionophore Is a compound that facilitates duffusion of an ion across a cell membrane.
A23187 Is a lipid soluble mol'c that enters the membrane and allows calcium to move across the membrane down it's conc. gradient.
DMSO stands for dimethylsulfoxide.
The key to a correctly controlled experiment is For any experimental condition, you should have another experimental cond. where not more than one variable changes btwn the 2 samples. Keeping sample sizes uniform and having enough replicates to make sure affects are not due to random variation in data.
MAPK lysis buffer with inhibitors lysis buffer lyses the cell membrane. The inhibitor contains protease inhibitors that stop MAKP from getting degraded. The others are phosphatase inhibitors that prevent dephosphorylation by stray phosphatases after the cells are lysed.
After MAPK is treated with lysis buffer and inhibitor cocktail, it is centrifuged. Where will the MAPK be? The supernatant. The pellet will contain nuclei and insoluble cell debris.
SDS-PAGE stands for sodium dodesyl sulfate
What is the role of SDS in electrophoresis? To eliminate the charge by coating all proteins with a net negative charge. It will also denature the proteins so that shape will not factor migration rate.
mercaptoehtanol is a reducing agent that breaks disulfide linkages (covalent bonds) in a protein
heat will remove any remaining folding not accomplished from SDS or mercaptoethanol.
The pre-cast gels contained 10% acrylamide. What is the fractioning range? 30-100kDa
The purpose of the stacking gel is? To compress all of the proteins in the sample into a thin band so that they enter the running gel simultaneously. This way proteins of the same MW will be present in discrete bands.
Want antibody will probe the sea urchin samples during SDS-PAGE gel? anti-pMAPK and anti-tMAPK
Why use Western Blotting? It is used when you need to specifically detect one protein in a mix of proteins.
How are the proteins transfered from the polyacrylimide gel to NC membrane? electrophoretically
Factors that influence protein transfer The buffer, the membrane, voltage, time and charge.
Why is voltage important when transferring a protein from polyacrylimide gel to a hydrophobic membrane like NC or PVDF? If the current is too high, low MW proteins could migrate too fast and not get a chance to bind to the membrane.
What does the blocking buffer consist of and what is it's purpose? It consists of TBST+5%BSA. It's purpose is to bind to unoccupied sites on the NC filter so antibodies won't stick non-specifically.
What does TBST stand for? Tris-Buffered Saline and Tween.
What does Tween do? Reduces non-specific binding of proteins to the antigens.
antigen The substances that antibodies bind to.
IgG Is a tetramer composed of 2 heavy chains and 2 light chains that are held together by disulfide bridges.
Constant Region The AA sequence in the base of the "Y". Does not vary for a given species.
Variable Region The AA sequence at the ends of the "Y". Does very and is the part of the IgG that bind to antigen. The diff AA seq. of the var. reg's in diff. IgG mol'cs creates spec. binding sites that bind to diff antigens.
What does it mean to raise antibodies against antibodies? Ab's raised against Ab's are secondary Ab's. Ex. using a goat to raise Ab's agains rabbit IgG's. Sec. Ab's bind to the constant region of primary antibodies.
The primary antibody is what binds to the... to the protein of interest.
The secondary allows you to... visualize the bound primary protein by producing a signal b/c it usually has a tag or peroxidase enzyme covalently linked to the con. reg.
How is the secondary Ab created? An animal is injected with the cons. reg. portion of IgG from the animal in which the primary Ab is raised.
How are Ab's helpful in biochem? They can be used to help determine where in a cell an antigen may be concentrated. Whether a cell express a certain mol'c on it's surface, whether a mol'c is present in a sample, or to quantify the amount of antigen in sample and for purifying an antigen
Is it possible for an antibody to bind an antigen other than the one for which it was created? Yes, some mol'cs have sim. structures. This is called cross reacting.
ELISA stands for Enzyme Linked Immuno Sorbent Assay
What does an ELISA accomplish? It is used to detect antigen-antibody complex with the help of an enzyme that converts a substrate to a colored product.
What antigen is detected by ELISA? inositol monophosphate, which is the breakdown product of a critical second messenger that is produced immediately after sperm bind (before Ca influx)
What are the three types of ELISA tests? antibody capture assay, two-site capture assay and competition assay.
What is the trigger for the cytoplasmic Ca influx in fertilized sea urchin eggs? The activation of PLC and the subsequent production of IP3.
What is PLC? It is a lipase that hydrolysis reactions on lipids to cut them into smaller pieces.
What does PLC do? It catalyzes the cleavage of the phosphorylated head group from a phospholipid called PIP2.
Where is PIP2 located? In the inner leaflet of the PM.
When PLC cleaves the IP3 head from PIP2, what is produced? Large quantities of IP3 and DAG.
What happens to IP3 when it is cleaved? It diffuses throughout the cytoplasm where it interacts with specific receptors. Receptor binding on the ER trigger Ca channels to open producing the Ca influx.
Why is LiCl added to the ELISA samples? To prevent IP1 from being broken down into free inositol.
What is the binding order in antibody capture assay? antigen->primary Ab->secondary Ab+covalently linked peroxidase or alkaline phosphate-> enzyme substrate(TMB)
What is the binding order in two site capture assay? Ab #1->antigen/sample->Ab #2-peroxidase conjugate->TMB
What is the binding order for competition assay? secondary Ab(anti-serum agst mouse IgG)->IP1-coupled w/peroxidase conjugate followed by monoclonal Ab against IP1(Primary Ab)->TMB
How does the competition assay work? The IP1 conjugate competes with the IP1 present in egg lysate samples to bind to a fixed # of primary Ab's present in well
In the ELISA test, what is the role of the peroxidase? It is used to produce a visible signal to detect Ab binding by continually converting substrate(TMB) to it's oxidized form, which amplifies the signal frm the binding Ab mol'c. This produces a signal such as color or luminescence.
Why is HRP used in ELISA? It is is used as as the peroxidase conjugate on secondary Ab, it catalyzes the oxidation reaction of peroxide and luminol.
What happens when TMB is oxidized? Shifts absorbance max giving a blue color.
Why did we add sulfuric acid in ELISA? To stop the oxidation rxn of TMB.
What does NBS stand for and what is it? nonspecific binding.is a negative control. some of the IP1peroxidase conjugate was added, but no primary antibody.the only signal would be from IP1-peroxidase conjugate that stuck "non-specifically" to the well, if for instance say it wasn't blocked well.
What was the total activity control for? It is a positive control. The control ensures that the peroxidase conjugate still has catalytic activity.
What is an epitope? The part of an antigen that is recognized by the Ab. Where the Ab binds. An antigen can have more than one epitope.
What are the 3 factors that determine how fast a molecule will migrate in the gel during electrophoresis? size, shape and charge
What 3 things ensure that all proteins loaded onto the gel are completely denatured in SDS-PAGE? SDS, mercaptoethanol, and heat
What produces the voltage across the electric field in electro phoresis? Give the rxn. Electrolysis (splitting of water with elctricity). Ions in solution(buffer salts) create the current. 4e- + 4H2O->2H2 +4OH (at cathode). 2H2O->O2+4H+4e-
Why did we had H2O2 to the substrate solution during ECL visualization? It is one of the rcts(substrates for peroxidase)
What two conditions must be met to illicit an immune response? antigen must be recognized as not self and the antigen must be large enough.
Created by: jriendea

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