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Adv.Biology > Labatory Techniques > Flashcards

Labatory Techniques Flashcards

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1
Q

What are the four techniques used to separate substances ?

A

Centrifugation
Protein electrophoresis
Iso-electric point
Paper,thin layer and affinity chromatography

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2
Q

A) what is a centrifuge ?

B) describe the process of centrifugation …

A

A) centrifuge is a piece of equipment that can spin a sample at very high speed

B) centrifugation is the process that allows substances to be separated according to their size and density

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3
Q

In the process of centrifugation, the largest and ______ materials separate out ____and form a ??

A

Densest
First
Pellet

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4
Q

The liquid which remains above the pellet is called the ….

A

Supernatant

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5
Q

Samples placed into a centrifuge should hold a similar volume and be placed in the rotor at ….

A

Opposite sides

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6
Q

Give factors that substances can be separated by …

A

Size, shape , charge and solubility

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7
Q

Based upon what factors does gel electrophoresis separate proteins ?

A

By their charge and or size / shape

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8
Q

How does gel electrophoresis separate proteins ?

A

Uses a current flowing through a buffer to separate the proteins.The gel used in protein electrophoresis acts as a sieve,separating the proteins

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9
Q

What happens during SDS - PAGE ?

A

During this procedure proteins are denatured and given a uniform negative charge which means the proteins can be separated based on their size as they migrate towards the positive electrode.Small proteins travel further through the gel than larger proteins

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10
Q

In gel electrophoresis , what can scientist analyse if the protein is not denatured ?

A

Allows scientist to analyse the proteins in their folded state

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11
Q

Antibodies can be used to detect both the ________ and ______ of a protein within a solution.

A

Presence and concentration

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12
Q

Briefly describe how the process of immunoassay works ….

A

This is a tech quite which uses antibodies linked to reporter enzymes that cause a colour change in the presence of a particular antigen.

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13
Q

Antibodies are used to detect the presence of a particular protein antigen within a tissue sample ….. What is is process known as ?

A

Immunohistochemistry

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14
Q

Antigens are used in a process caused protein _____.Proteins are _____ and transferred to a membrane which is probed for the protein of interest using a specific _____ that is linked to a detectable label

A

Blotting
Separated
Antibody

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15
Q

What is it essential that all animal cell culture posses ?

A

A media containing growth factors form SERUM

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16
Q

State each stage in the production of monoclonal antibodies …

A

1) a lab animal is exposed to the antigen of interest
2) B-lymphocytes are removed from the spleen of the animal
3) B-lymphocytes are fused with myeloma cells using polyethylene glycol,forming hybridomas
4) individual hybridomas are cultured in an multiwell plate
5) the culture medium can be removed and screened for the desired antibody
6) hybridomas which produce the desired antibody are cultured on a large scale

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17
Q

Which cell type in the mouse produces antibodies ?

A

B lymphocytes

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18
Q

What term is used to describe the fused cells

A

Hybridomas

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19
Q

Name the chemical used to make the cells fuse together

A

PEG/ polyethylene glycol

20
Q

What is the name given to an important aspect of health and safety which reduces the risk of hazardous materials coming into contact with the skin ?

A

Personal Protective Equipment : such as lab coat,safety glasses,gloves,long groused and closed toe shoes

21
Q

Give four examples of equipment used to measure liquids …

A

Syringe
Autopipette
Burette
Measuring cylinder

22
Q

What is the role of an auto pipette ?

A

To allow SMALL volumes of liquid to be measured VERY accurately

23
Q

What are burettes used to do ?

A

Dispense precise volumes of liquid reagents

24
Q

When measuring from a burettes it is important to take readings from the bottom of the ……

A

Meniscus - this is the curved top surface of the liquid inside the burette.

25
Q

Serial dilution involves ….

A

Reducing the concentration of a substance

in a solution repeatedly from a stock solution .

26
Q

State the definition of a colorimeter … and what they are used for …

A

A device that is used to measure the specific absorbance of a wavelength of light by a solution.They are used for quantifying the concentration of a pigmented compound.

27
Q

pH is a measure of the acidity or _____ of a solution.pH is a logarithmic measure of ____________ _____ concentration.

A

Basicity

Hydrogen Ion

28
Q

Buffers work by ……

A

Allowing the addition of hydrogen or hydroxide ions without affecting the pH of s solution.

29
Q

Describe what a standard curve is…

A

It is a type of graph that can be used to determine the concentration of an unknown solution and does this by using a series of standards of known concentrations that are measured and graphed.

30
Q

Give the definition of a haemocytometer and what it allows …

A

Is a device used to count cells and it allows an estimation of the concentration of cells in a sample to be made.

31
Q

Using the haemocytometer method,we can calculate the number of cells in each area do the grid using what formula …

A

1 X 1 X 0.1 = 0.1 mm3 of 0.1 ul .Also the number of cells counted needs to be times by the dilution factor which is 2 (1:1)

32
Q

If 15 cells were counted ,how do we calculate the number of cells per cm3

A

15 X dilution factor of 2 =30cells per 0.1ul
30 X 10 = 300 cells per ul
300 X 1000 = 300000 (3 X 10 5 ) cells per cm3

33
Q

What does the process of protein blotting involve ?

A

Antibodies are used to allow proteins from a tissue or cell extract to be detected .

34
Q

What is a hydriboma ?

A

This is formed when a myeloma cell fuses with antibody- producing B lymphocyte cell.

35
Q

State the 6 stages of the formation of monocle antibodies ….

A

1) a lab animal such as mice are exposed to the antigen of choice
2) B- lymphocytes are removed from the spleen of the animal
3) B lymphocytes are then fused with myeloma cells using polyethylene glycol,forming hybidomas
4) Individual hybidomas are cultured in a multi well plate
5) The culture medium can be removed and screened for the desired antibody
6) hybridomas which produce the desired antibody are cultured on a large scale

36
Q

What does the term ELISA describe ?

A

An analytical technique that uses antibodies to detect a substance in a solution by a colour change.

37
Q

What is the difference between indirect and direct ELISA?

A

In the indirect process,a secondary antibody is added to the multi well plate which is linked to the reporter enzyme and then binds to the primary antibody.

38
Q

In sandwich ELISA, what antibody is bound to the multi well plate ?
B) which antibody is bound to the reporter enzyme
C) what two antibodies are bound to the antigen

A

A) capture
B)secondary
C) captured and primary

39
Q

What is immunohistochemistry commonly used for ?

A

The identification of disease such as cancer as it can indenting abnormal gene expression.

40
Q

In immunohistochemistry, reporter enzymes are used to identity the abnormal tissue.
True or false ?

A

True

41
Q

What happens in the mobile and stationary stage of general chromatography ?

A

During the mobile phase the mixture is dissolved in a fluid and in the stationary phase components of the mixture are separated as they pass through.

42
Q

Describe what affinity chromatography is …

A

Proteins can be fractionated where by a mixture of proteins in solution is passed through a column containing a porous solid matrix

43
Q

Why are some proteins progression through the matrix slow?

A

As some proteins may have a greater affinity for the matrix material and therefore take longer to progress through the matrix after they are dissolved in a solvent.

44
Q

During paper chromatography where paper is used in the stationary phase what influences how far a substance travels along the solvent ?

A

The cellulose fibres in the paper are polar ,therefore the components of the proteins that are polar will bind to the paper quickly and won’t travel very far up the paper.

45
Q

The Rf value is expressed as a decimal fraction , what is the Rf value ?

A

This is the distance travelled by the amino acid from the origin divided by the distance travelled by the solvent from the origin

46
Q

How is Thin Layer Chromatography different from paper chromatography?

A

It uses silica gel rather than paper

Adv.Biology (7 decks)

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