QBM Lab 7

how low can the new method to visualize gels detect?

0.2 micrograms of protein on gel

what does the new method to visualize gels used/involve?

2,2,2-tricholoethanol (TCE) added to the separating gel solution during preparation, or soaked into the gel after the run is complete after electrophoresis the gel is careful removed from between the glass plates and placed in a GelDoc in the presence of UV light tryptophan residues in the protein bands on the gel react with TCE to produce visible fluorescence, which can be photographed

what system will be used in the lab?

Bio-Rad mini PROTEAN Tetra system

in this experiment what MW should GST-GFP and the controls have?

GST-GFP: 54kDa GST: 26kDa GFP: 27 kDa BSA: 66kDa lysozyme: 14 kDa

what do PVDF and blotting paper each do?

PVDF membrane has a high affinity for hydrophobic and charged amino acids blotting paper maintains the buffer and current

what is a molecular weight marker? what is its purpose?

a mixture of protein with exact molecular weights and often at specific concentrations loaded into one lane to serve as a reference to help determine the molecular weight and amount of the proteins in each band after separation

what is the SDS-PAGE gel matrix?

a polymer of acrylamide and N,N'-methylene-bisacrylamide (BIS), called polyacrylamide

how can protein concentrations be quantified by comparing their intensities?

a thin faint band contains less protein of that particular size than an intense bright band

what are protein gels made of?

acrylamide cast very thin in the vertical orientation

what are DNA and RNA cells typically made of?

agarose very thick and cast horizontally

what are markers and what are the also called?

also called standards or ladders loaded onto gels as a positive control to verify that the gel has run properly, and for estimating a proteins molecular weight and concentration

in an ideal purification, what will samples that are properly normalized show?

an increasing intensity of the target protein and a decreasing intensity of contaminant bands

what charges do the anode and cathode have in an electrolytic cell?

anode: positive cathode: negative

what does the tracking dye do?

assists in loading the samples (since they will now be blue), its movement verifies current is passing though the gel, and since it runs faster than proteins, it can be used to determine when the current should be stopped at the end of the run

why are acrylamide gels cast vertically?

because they are very thin, so that when samples are loaded they fall to the bottom of the wells

what does B-ME do once added to the protein?

breaks disulfide bridges which are covalent

what does SDS do when added to the protein?

breaks weak bonds and denatures proteins to their primary structure

how will relative purity of GST-GFP be determined?

by analyzing the intensity of GST-GFP in each sample and the number of bands in each lane

how is electrophoresis performed?

by attaching electrodes from a power supply to the running module holding the gels

what does the current from electrophoresis cause the proteins to do?

causes the proteins to move downward towards the positive electrode (anode)

how can protein sizes be estimated in SDS-PAGE?

comparing the distance raveled to the control bands in the molecular weight marker, or by their relative mobility on the gel, which is the ratio of how far the protein travels in comparison to the tracking dye

what doe makers contain and what are they used for?

contain proteins of known molecular weigh which on be compared to the protein in the sample lanes

what dye can be used for visualization of the proteins after SDS-PAGE is completed? why?

coomassie brilliant blue R-250 dye it has a strong affinity for proteins

in this experiment why would detection via enzyme assay or fluorometry need to be performed?

due to denaturation, SDS-PAGe does not indicate whether proteins in a source sample were properly folded and functional

what makes up the western transfer apparatus and what happens?

electrodes are attached to this assembly, and an electric current is passed through the membrane-gel sandwich perpendicular to the direction of the gel as proteins exit the gel, they are immobilized on the membrane in the same relative positions as they were on the SDS-PAGE gel

T/F agarose gels require a stacking gel

false

T/F the protein samples are boiled before the sample loading buffer is added

false boiled after the loading buffer is added

T/F the migration through the gel is exponential

false logarithmic

T/F once SDS is added to the protein, the proteins can migrate to the negative electrode to be separated sole based on size

false migrate to positive electrode

T/F TCE is protein specific

false not protein specific

T/D B-ME is an oxidizing agent

false reducing agent

how are the bands made visible, what is used? (After removal from glass plate)

fixing them to the gel with methanol and acetic acid to prevent dispersion, staining with Coomassie and destaining with methanol, after which protein bands will appear as blue bands on clear gel

where is the membrane gel sandwich placed?

in a buffer filled chamber or onto a semi dry western transfer apparatus

the polymerization of acrylamide and BIS is induced by/involves what components?

induced by free radicals, contributed by ammonium per sulfate (APS), and the reaction is catalyzed by tetramethylethylenediamine (TEMED) which acts as a free radical stabilizer

why is agarose cast between two plates?

it can be cast in an open tray but oxygen inhibits acrylamide polymerization creates a smooth gel interface between the stacking and resolving gels

after visualization on the GelDoc what happens to the gel?

it is sandwiched between a nitrocellulose or polyvinylidene fluoride (PVDF) membrane and blotting paper

what can be used for the highest sensitivity to visualize bands?

label with a radioactive molecule and deter with autoradiography

how are protein samples loaded in SDS-PAGE? what happens as a result?

loaded on top of the gel and in the direction of the gel and current as a result, if current were passed through this gel, the proteins would enter the gel as a thick band and not be correctly resolved

what does SDS coat the protein in? what does this do?

negative charges provides proteins with equal charge to mass ratio allowing them to migrate toward the positive electrode and separate based on size

when is the comb removed from the gel? what does this create?

once the stacking gel has solidified creates wells into which proteins can be loaded

when is the comb inserted into the stacking gel?

once the stacking gel is poured on top of the solidified separating gel and prior to polymerization

what pH does the stacking gel have what does this ensure?

pH 6.8 ensures the proteins concentrate between Cl- and glycine in the buffer

what pH does the resolving gel have and what happens as they enter it?

pH 8.8 glycine becomes negatively charged, allowing the proteins to carry the current and be separated by size

what does the band intensity mean and how can it be quantified?

protein amount comparing them to reference bands in the marker

how much acrylamide is in the separating/resolving gel? in the stacking gel?

resolving: 8-25% stacking: 5%

prior to SDS-PAGE separation what are the protein samples mixed with?

sample loading buffer, which contains sodium dodecyl sulfate (SDS), B-mercaptoethanol (B-ME), glycerol and a tracking dye (bromophenol blue)

in SDS-PAGE which proteins migrate more quickly and where do they appear?

smaller proteins migrate quickly, and appear as bad at the bottom of the gel

what does SDS-PAGE stand for?

sodium dodecyl sulfate polyacrylamide gel electrophoresis

which gel is porous and what do the pores do?

stacking gel allows proteins to travel quickly and concentrate into a tight band before reaching the resolving gel, where they are separated by size

what structures to proteins form in their native state?

tertiary, three dimensional structures some can form quaternary

what can be analyzed and what can be quantified from an SDS-PAGE?

the bands can be visualized and analyzed to determine the molecular weights of the proteins, and the band intensities can be quantified to determine the relative amounts to the protein in the sample

how are the proteins location and quantity determined after SDS-PAGE has been completed?

the gel is removed from the glass plates, placed in a container, fixed, and stained with gel staining solution

after the western transfer, what can be done to the membrane?

the membrane can be probed with an antibody specific target protein to interest in a technique called western blotting

how is the gel of SDS-PAGE cast?

the mixture is pipetted between two glass plates separated by 0.75 or 1mm spacers

what is one advantage of using TCE to visualize gels?

the protein bands are not fixed to the gel which can be removed from the GelDoc and used in subsequent applications

when the electric current passes across the gel, what happens to the proteins?

the proteins migrate towards the positive electrode at a rate that corresponds to their frictional resistance to the gel matrix

what is electrophoresis?

the separation of charged molecules by a current

where do larger proteins appear on the gel in SDS-PAGE? why?

the top of the gel they migrate more slowly because they don't fit as easily though the pores of the gel

what is the lane?

the vertical space in the gel below each well

what is the benefit of a thin gel?

thin cells allow for easier staining and transfer and prevent the gel from overheating

after visualizing the gel and taking a picture the proteins will be transferred to what and how?

to a membrane via western transfer

why must proteins be linearized from their tertiary/quaternary structures?

to be separated by size alone on a gel

in this experiment what will SDS-PAGE and TCE be used for? how?

to verify the presence of GST-GFP in the lysate, affinity and IEX samples by comparing its MW to markers

what are proteins treated with in SDS-PAGE? what happens to the proteins?

treated with SDS, which gives them a negative charge loaded into the wells of a gel

T/F SDS acts as a detergent

true

T/F SDS is also added to the gel and buffers to keep the samples denature through the separation

true

T/F because TCE is not protein specific, a single band may not indicate a pure protein, as two proteins may have the same molecular weight

true

T/F the gel is first placed into a buffer-filled tank and then the samples are loaded into the top in the direction of the gel

true

in SDS-PAGE, how do we ensure proteins migrate as a high band through the gel?

two gels are cast separating (resolving) gel on bottom, which has a high percentage of acrylamide (8-25%) and a stacking gel on top, with a low percentage of acrylamide (5%)

what does SDS-PAGE use to separate proteins and what does separate proteins by?

uses a gel matrix to separate proteins by size

what will a western transfer prepare the samples for?

western blotting

when is electrophoresis complete?

when the tracking dye reaches the bottom of the gel