Proteins lab final

Why do you tare a balance? What extra step must you do if you choose not to tare?

zero it otherwise subtract the amount of just the weight boat

Calculations necessary for sample/buffer dilutions

C1V1\=C2V2

how would you prepare a dilution of 400 mL of a 1X solution from a 5X stock?

(5)(x) \= (1)(400)

x \= 8ml

400-8\=392 mL of water

From a 10X stock?

(10)(x) \= (1)(400)

x \= 4ml

400-4\=396 mL of water

From a 20X stock?

(20)(x) \= (1)(400)

x \= 2ml

400-2\=398 mL of water

Molarity

mole of solute in solution/ liters of solution

Percent Composition

(molar mass of all elements in g/mol) / (molar mass of entire compound in g/mol) *100

Prepare a dilution in a single step such as with the antibodies?

put some from one tube to the next, to the next, to the next

prepare 25 mL of a 1:4000 dilution?

25 mL/ 4000 \= .00625ml

X1000\= 6.25 mircoL

prepare 25 mL Of a 1:7000 dilution?

25/7000\=.00357ml

x1000\=3.57mircoL

prepare 25 mL Of a 1:24,000 dilution?

25/24000\=.001ml

x1000\=1.04

Pipet limitations/Do you know which one to use for a specific volume/Do you know how to read them?

P20,P200,P1000

Can you convert between mL and μL

ml to μL just X 1000

precision

how close measurements are to each other

accuracy

how close a measurement is to the true value

how to calculate the pipetting error

(mean volume - intended volume) / (intended volume) ) X 100 \=

Parts of a light microscope?

Light Microscopy

use of any kind of microscope that uses visible light to observe specimens

stains used in this lab - L, N, and C1 or C10

stain 1 dipped twice, drain dip into stain 2 twice, rinse w dH20, airdry

would bacteria be classified as prokaryotic or eukaryotic

prokaryotic

staphylococcus

cluster of cocci

Bacillus cereus

rods

colchicine morphology effect

cytotoxin that inters w/ mircotubule polymerization

-multiple nuclei

-membrane blebbing

-giantism

large cytoplasmic vacuoles

-breakdown of structural integrity

With the resolution of the light microscopes used in this lab, what organelles were you really able to see with the bacteria?

With the resolution of the light microscopes used in this lab, what organelles were you really able to see with the animal cells?

mobile

Spirostomum,

Didinium

Tetrahymena

immobile

Clostridium

Volvox

Rotifera

Micrasterias

which feature allowed for mobility?

cilia, flagella

Were they prokaryotic or eukaryotic

eukaryotic

Were they photosynthetic or not

mircosterias

Closterium

How would you microscopically determine being photosynthetic?

green

Tumor vs normal cell characteristics \-- how are the cells shaped differently? What are the differences in the growth patterns between these cells?

cancer cells have a variation of cell shapes, more oblong, rigid, larger abnormal nucleus.

normal cells are more uniform in size and shape

Tumor vs normal cell: What are the differences in the growth patterns between these cells?

cancerous cells grow & divide rapidly, tend to not have clear cell boundaries

What is a homogenate

a suspension of cell fragments and constituents that is obtained with a tissue is homogenized.

Why is 0.25M sucrose used for homogenization?

isotonic buffer that allows the cells to be further broken down.

mimics the same osmotic pressure as in vivo

Why are tubes balanced in the centrifuge? How does the size of molecules determine how they pellet during centrifugation

centrifuges spin at extremely high speeds to separate material. And an unbalanced centrifuge will not operate at peak efficiency. In fact, diminished product quality or inconsistent product output can be an indicator of an unbalanced bowl.

what features of a molecule determine whether it will pellet first in a centrifugation spin? Why?

size and density

the centrifugation chart

to find rpm & gravity

How does the Bradford dye work?

colorimetric dye

binds primarily to basic aa residue

blues vary

BSA

bovine serum albumin

why is BSA used?

used as known standard protein concentration @ 2mg/ml

draw a standard curve?

-plot concentrations of diluted samples vs ABs

-find question and turn on R2

-plug ABs into y to find x (diluted samples)

serial dilution

A stepwise dilution of a substance in solution

what is Acrylamide used for?

native-page: nonnaturing, separates by size, shape, charge

sds-page: denaturing, separates by size

bis-acrylamide used for

polymerization of gel

agarose used for

makes pores

the higher the concentration of monomers,

the smaller the pores are

Coomassie blue stain

dye that stains proteins blue and allows them to be visualized

ponceau S stain

detection of protein bands on Western blot membrane

What information about proteins can be determined by SDS-PAGE?

size. Separate proteins on the basis of their size (molecular mass) . It is an anionic detergent that unfolds proteins and gives them a negative charge. It is a denaturing gel system for proteins. Breaks disulfide bonds

How did you obtain the data to generate the standard curve?

found distances of bands compared to the marker proteins (Rf)

graph Rf vs log MW of the given marker proteins to find equation

plug in found Rf into x, solve for new log MW value

do anti-log to find MW

What is the difference between this curve and the one for the protein determination by Bradford?

this curve shows Rf vs logMW

other one shows diluted samples vs ABs

SDS

anionic detergent, coats with -

BME

breaks disulfide bonds

Rf

distance traveled by protein/distance travel by marker

logMW \=

-m(Rf) +b