Laboratory Techniques Bio and Chem Flashcards

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1
Q

In general when doing Western blotting the ____ will migrate down quickly through the gel

A

Shortest amino acid sequence have lower molecular weight and migrate down quickly through the gel

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2
Q

___ can detect and quantify proteins.

A

Enzyme Linked immunosorbant Assay (ELISA)

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3
Q

How does ELISA work?

A

A primary antibody (linked to a “reporter” enzyme) is added, which binds the antigen (protein). Samples are then washed to remove unbound proteins, and the reporter enzyme substrate is added. Enzyme-substrate reaction creates product that results in a quantifiable/detectable signal

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4
Q

__ is used to analyze mRNA expression levels

A

Northern Blot

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5
Q

__ is used to detect proteins

A

Western Blot

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6
Q

__ is used to separate proteins by molecular weight

A

SDS-Page

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7
Q

What are the steps of SDS-Page

A

SDS coats proteins with a negative charge. An electric current is then applied, smaller proteins travel through the polyacrylamide gel toward the POSITIVE anode faster than larger ones, creating lanes of size separated protein bands

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8
Q

___ is a technique used to separate liquid molecules based on their boiling points, compounds with the LOWEST boiling point evaporate first

A

Distillation

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9
Q

When would you use simple distillation, fractional distillation, or vacuum distillation?

A

Simple distillation : bp<150 C and >25C
Fractional distillation: bp <25C
Vacuum Distillation: bp> 150C

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10
Q

___ separates molecules based on boiling point.
Intermolecular forces, molecular weight, and branching all affect boiling point. For compounds with same functional group but different carbon counts, higher molecular weight equates to a higher boiling point and longer retention time because of strong interaction with liquid phase

A

Gas-liquid chromatography

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11
Q

___ is a technique that separates components of a mixture based on polarity, polar molecules gave a higher affinity for the plate and travel up smaller distance than nonpolar molecules

A

Thin Layer Chromatography

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12
Q

___ is a type of chromatography in which a liquid solvent (mobile phase) carries the sample through a column filled with adsorbent material (stationary phase)

Molecules with polarity similar to the stationary phase have longer retention time than molecules with polarities similar to the mobile phase

A

High Performance liquid chromatography (HPLC)

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13
Q

A biochemical technique that utilizes antigen-antibody interactions to determine the presence of either

  • antigens (proteins or cytokines)
  • Specific immunoglobulins (antibodies)
A

Enzyme-linked immuno-sorbent assay (ELISA)

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14
Q

give the 6 steps of ELISA?

A
  1. Experimental wells are coated with antibodies for target antigen
  2. Sample of serum or cell extract is added to wells
  3. Antibodies immobilize the antigen by binding to it
  4. Any unbound proteins remaining in sample are washed away
  5. Enzyme-linked antibody that also recognizes target proteins are added to wells
  6. The wells are filled with a solution that changes color in presence of the detection enzyme.
    A color change indicates target protein was present in sample
    No color change means protein was absent
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15
Q

___ are used more extensively in the medical field to measure the relative amounts of hormones or drugs in patients’ serum

A

Radioimmunoassay (RIA)

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16
Q

5 steps of Radioimmunoassay

A

Step 1. A known amount of radiolabeled antigen (example: insulin that was synthesized with Iodine 25-labeled tyrosines) is incubated with a known amount of antibody that is specific to that antigen
Step 2. The insulin:antibody complexes are isolated
Step 3. The total amount of radioactivity is measured
Step 4. Unlabeled insulin is mixed into the solution in increasing amounts. The unlabeled insulin competes with the labeled insulin for the antibody. As more unlabeled insulin is added, less total radioactivity is recovered and measured. This competition assay helps formulate a standard curve.
Step 5. Steps 1-3 are repeated using patient serum instead of the unlabeled insulin. The standard curve is used to extrapolate the amount of insulin that is circulating in a patient’s serum

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17
Q

___ is a means of separating things (nucleic acids, proteins, etc.) by size or by charge.

A

Electrophoresis

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18
Q

How do things in Gel Electrophoresis move?

A

Things move through the gel toward positive pole, according to size; smaller things migrate faster (they fit more easily through the pores of the gel) and larger things migrate more slower)

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19
Q

This enzyme is used in Gel Electrophoresis to cleave the strands of DNA into smaller fragments of varying size

A

Restriction endonucleases

20
Q

___ allows you to detect the presence of specific sequences within a heterogeneous sample of DNA. This process allows you to isolate and purify target sequences of DNA for further study

A

Southern Blotting

21
Q

What are the 3 steps of Southern Blotting?

A
  1. Separate DNA fragments on an electrophoresis gel
  2. Transfer fragments to a nitrocellulose membrane
  3. Filter is “probed” for the target DNA sequence. Hybridization probes are short single-stranded sequences of nucleic acid (usually DNA) that have two important features:
    - complementary to a portion of target DNA sequence
    - constructed with radiolabeled nucleotides, which allow the visualization of the target sequence with special film
22
Q

___allows you to detect the presence of specific sequences within a sample of RNA

A

Northern Blotting

23
Q

___allows you to detect the presence of certain proteins within a sample and serves as a diagnostic tool

A

Western Blotting

24
Q

In Western Blotting, the smallest proteins will migrate__

A

fartehst from the wells because the proteins are negatively charged

25
Q

__blotting is used to analyze post-translational modifications of peptides, such as the addition of lipids or carbohydrates

A

Eastern Blotting

26
Q

___ is a protein that has been obtained by transcribing and translating a novel combination of DNA (recombinant DNA) from different organisms

A

Recombinant protein

27
Q

___ cut nucleotides from the ends of DNA chains

A

Exonucleases

28
Q

____ cuts in the middle of the DNA chain

A

Endonucleases

29
Q

What is the natural purpose of endonucleases in bacteria?

A

Their natural role in bacteria is to destroy viral DNA that gets injected into the cells, they RESTRICT reproduction of hostile viruses

30
Q

Sequences of DNA with two fold rotational symmetry (the nucleotides in the 5’ direction are the same as those in the 3’ direction) are known as __

A

Palindromes

31
Q

Many restriction enzymes recognize ___

A

Palindromic sequences

32
Q

The solubility of solids in liquids tends to increase with __

A

increasing temperature

33
Q

The solubility of gases in liquids tends to ___ with increasing temperature

A

DECREASE WITH INCREASING TEMPERATURE

34
Q

The solubility of gases in liquids tends to ___ with increasing pressure

A

INCREASE WITH INCREASING PRESSURE

35
Q

What is the natural role of restriction endonucleases?

A

They are used by bacteria to destroy viral DNA that gets injected into the cell

36
Q

___ is a very quick and inexpensive method for detecting and amplifying specific DNA sequences, screening hereditary and infectious diseases, cloning genes, and fingerprinting DNA

A

Polymerase chain reaction (PCR)

37
Q

A competitive inhibitor will_____

A

INCREASE the km and Vmax is unchanged

38
Q

The pitch of a perceived sound is dependent on its__

A

frequency

39
Q

What are the order of relative reactivities of carboxylic acid derivatives?

A

Acyl halide > anhydrides > esters and acids > amides

40
Q

Sound travels fastest in __ and slowest in __

A

Sound travels fastest in solids and slowest in gases

41
Q

Sound waves moving through two equally stiff solids (bulk modulus) will travel fastest in the ____ material

A

LESS DENSE

42
Q

The presence of __ at the neuromuscular junction is required for skeletal muscle contraction

A

Acetylcholine

43
Q

__ separates molecules based on affinity

A

Gas chromatography

44
Q

__ separates particles by density.

More dense particles gravitate towards bottom of spun tube.

Less dense substances remain at top in the liquid supernatant

A

Centrifugation

45
Q

Nucleophilicity decreases from ___ on periodic table

A

Left to Right

46
Q

___ is part of the bodies adaptive immune response, it is provided by B cell activity which promotes an antibody or immunoglobulin

A

Hummoral immunity