lab shizzle

Why is DTT in the Laemmli sample Buffer

Breaks disulfide bonds by reduction to destroy tertiary protein structures

The secondary antibody was conjugated with HRP, what does that stand for?

Horseradish Peroxidase

Name one type of posttranslational modification

Proteolytic Cleavage

What is the term used for laboratory methods that use antibodies to detect proteins

Immunodetection

How does HRP work in our experiment

The primary antibody interacts with HRP, producing a purple/gray precipitate that deposits color(?)

What is immunology

the study of the immune system and how the body protects itself against disease

what is the target of the primary antibody used in the experiment

Myosin light chain

Proteomes differ from cell to cell

true

What effect does heating the sample have on the extracted material

Heat helps to denature the proteins

What is any foreign invader that elicits antibody production

Antigen

Describe how to make an antibody to detect another muscle protein such as dystrophin

Inject Distrophin antigen into animal (e.g goat). The animal model will produce a primary antibody. Purify/extrat the primary antibody, then inject into a secondary animal model. Purify extract the secondary antibody the second animal forms. Tag the secondary antibody from the second animal with a colorimetric substrate, like HRP in the experiment. Add primary antibody to the nitrocellulose membrane containing distrophin, wash, add secondary, wash, add enzyme/substrate that reacts with colorimetric tag on secondary antibody, which will detect the distrophin.

What is the secondary structure of a protein

The local folding patterns of the polypeptide chain, like alpha-helices and beta-pleated sheets

What are the common names of the 5 fish we are studying in the Protein Profiler experiment (written)

Swordfish, Salmon, Trout, Tuna, Cod

There were two control lanes on your protein gel, choose both

Presion Plus Protein Kaleidoscope standards, Actin and Myosin Standards(?)

One Dalton is defined as

the mass of a hydrogen atom

what is the role of glycerol in the Sample Buffer

to make samples sink into wells

what is the primary structure of protein

the linear sequence of amino acids in a polypeptide chain

What is the quarternary structure of a protein

The arrangement of multiple polypeptide chains (subunits) to form a functional protein

What is the charge on a nitrocellulose membrane used in Western Blotting?

Positive

Why are proteins treated with ionic detergent (SDS), reducin agents (DTT), and heat before SDS-PAGE?

SDS, DTT, and heat denature proteins,
SDS confers a negative charge to the proteins

Multiple polypeptides can combine to form complex structures such as the muscle protein myosin. How many polypeptide chains are in the muscle protein myosin?

4

Why is a polyacrrylamide gel used for separating proteins

it has a smaller pore size than agarose, ideal for separating proteins that are much smaller than DNA

What is an immortalized antibody producing gell

Hybridoma

What is the tertiary structure of a protein

the overall three-dimensional shape of a single polypeptide chain, which is determined by interactions between the side-chains (R-groups) of the amino acids

What type of chemical bond holds the heavy and light chains of an antibody together

Disulfide bonds

What is proteomics

The study of proteins, their structure, function, and interactions

why is it important to denature proteins before electrophoresis?

Denaturing linearizes proteins. Linear proteins can more readily migrate throough the gel matrix and be seoarated according to protein mass.

What is the purpose of blocking in a Western blotting protocol?

Blocking is used to prevent non-specific binding of antibodies to the membrane

What is the colorimetric substrate used in this lab

4CN

Why are proteins from SDS-PAGE gel transferred to a nitrocellulose membrane (Western blotting?)

Proteins bound to the surface of a membrane are more accessible to antibodies
Without blotting, proteins remain within a flimsy and fragile gel
A membrane is more stable and longer lasting than a gel

Which of thses is NOT Posttranscriptional Modification

DNA Methylatioon

What is the purpose of Western Blotting?

Western Blotting is used to identify and analyze secific proteins in a sample by using antibodies to detect them

The molecular mass of myosin light chain is approximately 22 kD, myosin heavy chain is 200 kD, and actin is 42 kD. Which protein will migrate the fastest through the gel?

myosin light chain

Which is most likely the correcct ordering of the natural selection process?

Mutation-->Variation-->Specialization-->Speciation-->Evolution