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Biochemistry 3380 > Exam 1 > Flashcards

Exam 1 Flashcards

1
Q

What are the 6 common units of concentration?

A

M; % w/v; w/v; % v/v; v/v; X

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2
Q

What does qs stand for?

A

quantity sufficient

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3
Q

What does BTV stand for?

A

bring to volume

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4
Q

% w/v is used when

A

the pure solute can be commonly found as a solid

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5
Q

What does it mean to have a 9% w/v solution?

A

You have 9 g of solute per 100 mL solution

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6
Q

When is w/v used?

A

commonly used ratio for dyes and protein mixtures

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7
Q

When is % v/v used?

A

used when the pure solute can be commonly found as a liquid

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8
Q

What does it mean to have a 70% v/v solution?

A

70 mL solute per 100 mL solution

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9
Q

Where do we see v/v?

A

in older cell biology literature/protocols

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10
Q

v/v refers to

A

parts - volumes aren’t given, just the relative ratios of each component

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11
Q

What are the units of X?

A

unitless

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12
Q

The ____ solution is always more concentrated than the ____ solution.

A

The stock solution is always more concentrated than the working solution.

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13
Q

The working solution concentration is always

A

1X, unless told otherwise

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14
Q

What is the dilution formula?

A

C1 V1 = C2 V2

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15
Q

When is the dilution formula commonly used?

A

when making a solution from a chemical stock

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16
Q

What is a serial dilution?

A

series of solutions that are all diluted by the same factor

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17
Q

What is an independent dilution series?

A

different dilution factors in the series

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18
Q

What is the physiological role of phosphatases?

A

to remove phosphate groups which become available for metabolic activities like ATP and nucleotide synthesis, phospholipid synthesis, and regulation of metabolism

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19
Q

What are the two main groups of phosphatases and how are they classified?

A

alkaline and acidic; classified by pH optima

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20
Q

What is the basis of the reaction to detect the presence of phosphatase activity?

A

Add PPP, then sodium carbonate to stop the reaction and alter the structure of PPP, causing it to turn pink

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21
Q

What is the purpose of sodium carbonate?

A

to stop the reaction

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22
Q

What does it mean when a protein is either heat stable or heat labile?

A

heat stable means it is not affected by temperature changes; heat labile means it is affected by temperature changes which implies it has a temperature optimum

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23
Q

Why is it important to consider the application of a purified protein prior to designing a purification scheme?

A

You would like to know what you want to optimize - either activity, presence, or purity

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24
Q

What are the four intellectual steps to optimizing the purification of a protein?

A

development of a suitable assay, selection of the best source material, solubilization of the desired protein, development of a suitable series of fractional steps

25
Q

Why should you never follow a seal act with a seal act?

A

Don’t follow a separation procedure with another one that achieves separation based on the same property. This is because contaminating proteins that co-purify with the protein of interest in the first separation are also likely to co-purify with the desired protein in the second separation.

26
Q

When might you use a coupled reaction?

A

When you are unable to detect the product of the primary reaction and must change the product to see it.

27
Q

When might you use a continuous time assay?

A

When you want to know all of the data points instead of just taking measurements intermittently

28
Q

The darker the pink, the

A

more the activity

29
Q

What is the purpose of the 30 minute incubation following the addition of PPP?

A

removes the phosphate groups

30
Q

How does Na2CO3 work?

A

denaturing the enzyme and alters the structure of PPP, turning it pink

31
Q

What is GFP?

A

green fluorescent protein, found naturally in the medusa of aequorea victoria

32
Q

Why does GFP glow green?

A

fluoresces due to its excitation wavelength of 395 nm and its emission wavelength of 510 nm, producing the color we see as green

33
Q

Why was the GFP(uv) gene placed into the pRSETA plasmid?

A

this was so that it can easily be placed into a bacterial strain of your choice

34
Q

How is a calculated MW and an estimated MW of a protein determined?

A

calculated - determined by gene sequencing, finding exact amino acids, and adding known MWs of each AA; estimated - determined by number of Das and using the average AA weight which is 110-120 Da

35
Q

Why was the pRSETA-GFP(uv) transformed into the E coli strain BL21pLysS?

A

We treated the strain as a search engine as well as an amplifier; the BL21 would take the protein and over-express so that it is easier to detect

36
Q

What is the general process of inducing expression of rGFP?

A

IPTG inhibits the Lac repressor, allowing the lac promoter to run, which leads to the production of T7 RNA polymerase. This then is able to overcome the lysozyme inhibition and bind to the T7 promoter region, allowing the rGFP gene to be expressed

37
Q

Why was the GFP placed under the control of a T7 promoter?

A

any gene downstream of the T7 promoter is inducible

38
Q

What is the purpose of adding IPTG to the growing culture?

A

It inhibits the Lac repressor, which allows for the transcription and expression of the rGFP gene

39
Q

What was the purpose of the pLysS plasmid?

A

the lysozyme inhibits the T7 RNA which prevents T7 from binding with the promoter. Lysozme also degrades the cell walls of neighboring bacteria during the quick thaw.

40
Q

What was the purpose of adding ampicillin and chloramphenicol to the media?

A

helped eliminate contaminating bacteria in the culture and isolated the E coli grown with the antibiotic-resistant plasmid

41
Q

Why do we have to take culture samples before and after the addition of IPTG?

A

In order to have a control group with which we can compare the pre-induction fluorescence to the post-induction fluorescence. G0 could have auto-fluorescence because of contaminants.

42
Q

Who first discovered GFP?

A

Shimomura

43
Q

Who first cloned GFP?

A

Prasher

44
Q

What was the first transgenic organism to express GFP?

A

E coli

45
Q

What was the first transgenic eukaryote to express GFP?

A

C elegans

46
Q

Who reported the expression of GFP in heterologous systems?

A

Chalfie et al

47
Q

What is the relative molecular weight of GFP?

A

27 kDa

48
Q

The open reading frame codes for a

A

238 amino acid protein

49
Q

The chromophore is

A

protected inside of an 11 stranded beta-barrel

50
Q

Why is the GFP protein unique in terms of stability?

A

stabile in changes in oxidation/reduction; chemical reagents; pH; temperature

51
Q

Why is GFP a valuable research tool? (6)

A

no cofactor required for fluorescence; detection is easy and cheap; immediate detection is possible; small protein - can be used as fusion tag; dual labeling/detection; expression in heterologous systems possible

52
Q

What is the wild type?

A

the version found in nature

53
Q

How is GFP uv different from GFP?

A

GFP uv has additional amino acids, including a His-6 tag

54
Q

What is the V strain?

A

vector strain, contains everything EXCEPT the gene of interest (GFP-uv)

55
Q

What is the excitation wavelength for GFPuv?

A

395 nm

56
Q

What is the excitation wavelength?

A

wavelength used to excite electrons and cause fluorescence

57
Q

What is the emission wavelength for GFPuv?

A

510 nm

58
Q

What is the emission wavelength?

A

wavelength detected from excitation process (wavelength we see)

59
Q

Draw out slide 39.

A

draw

Biochemistry 3380 (9 decks)

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