Cell culture and an overview of techniques used to isolate and visualise proteins

Question 1

what is cell culture?

Answer 1

• Cell culture refers to the removal of cells from an animal or plant and the process by these are grown in a favourable artificial environment- controlled conditions, generally outside their natural environment

Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of:
• Suitable vessel (flask or dish or well that cells grow in) containing a substrate or
• Medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones etc.
• Incubator (humid CO2 incubator at the appropriate temperature)

Question 2

what is the difference beween adherent cells and non-adherent cells?

Answer 2

Non adherent cells: suspension cells are suspended in liquid as single cells or free floating clumps o.e. can move around

Adherent cells: grow in a single layer called a monolayer. Cell growth is limited by the surface area i.e. they stick to the plastic of the flask and can’t grow beyond the flask= can get overcrowded.

Can lift adherent cells/ enzymatically detaching the cells from the plastic i.e. cells are now non-adherent and can float in the cell culture medium instead of being stuck to a surface).

Question 3

what do we mean by density?

Answer 3

Density is a good measure on how well cells are proliferating.

Cells are grown and maintained using the appropriate conditions. When they get too dense/ cells are overcrowded for the flask= they become stressed= have to remove some of the cells= it must be passaged (sub-cultured).

Question 4

define passage number

Answer 4

Passage number is the number of times a cell culture has been sub-cultured (some cell lines can only be grown for a finite number of passages). Cells often grow finitely= don’t tend to grow cells beyond passage 20 as they get too old.

Question 5

what do we mean by confluency?

Answer 5

Cell density can be a measure of proliferation. It is usually combined with an estimated (or counted) percentage, so 10% confluency means that 10% of the surface the dish or flask used is covered with cells, 100% means that it is entirely covered. In a normal environment (within the lab), we want a 30-50% confluency so they can double overnight. Any more than that; they start to act differently.

Question 6

what is cell passage (sub-culture)?

Answer 6

Involves lifting the cells, diluting the volume and removing some of the cells.

Question 7

How can cells be released or detached from the flask/dish?

Answer 7

Cells may be released or detached from the flask/dish enzymatically (trypsin EDTA) or mechanically.

Enzymatically: Trypsin cleaves peptide bonds in fibronectin of the extracellular matrix (that cause the cells to attach to the plastic). EDTA chelates/binds to calcium ions in the media that would normally inhibit trypsin

• Trypsinizing too long will reduce cell viability
• Mechanically: Cell scraper

Question 8

how can cells be quantified?

Answer 8

• Cells can be quantified by counting a sample in a haemocytometer using a counter to determine cell density.

• 4 outer squares are important
• Use a formula to work out how many cells you have per ml

Each chamber of a hemocytometer is ruled with nine major squares. When filled and cover slipped, each square (shown in red) is 1.0 x 10-4 ml.

EXAMPLE:

Sample
The following numbers of cells were counted across two of the squares of the chamber: 34, 37, 29, 38, and 32. Calculate the cells/ml.

Answer:
Average cell count (34, 37, 29, 38, 32) / 5 = 34
Average cell count in one square 34 /2 =17
Therefore there are 17 cells in 1.0 x 10-4ml
Or 17 x 104 cells/ml (or 1.7 cells x 105cells/ml)

Question 9

what occurs once cell number/ cell quantification is determined?

Answer 9

• Seeding cells for e.g. multi well assay or seed cells in flasks to use in growth curves
• E.g. if you are treating cells with different drugs= can make a direct comparison between wells (As same number of cells).

Question 10

what can use the cells from the seeded experiments for?

Answer 10

• Can use cells from seeded experiments to make protein extracts
• Many ways to treat cells e.g. use DNA damaging reagents, chemotherapy etc.

PROTEIN PURIFICATION/ISOLATED AND ANALYSED:

• Whole cell extract
• Cytoplasmic extract
• Nuclear extract

Question 11

• Trypsin can’t be used for protein extraction; it interferes with the phosphorylation state of proteins on the cell surface. Thus, it is better to mechanically lift cells–>

Answer 11

Harvesting Cells:
Mechanically lifting cells- cell scraper
1) Remove media from cells
2) Harvest cells by scraping cells from flasks/dishes etc into the phosphate-buffered saline (e.g. PBS)
3) Centrifuge to get a cell pellet= use to extract protein

Question 12

Describe protein synthesis

Answer 12

• Crucial for cell growth, proliferation and survival
• Expensive process for the cell, therefore tightly regulated
• Regulation can control overall rates of protein synthesis and modulate the expression of specific transcripts

Regulation can be at 2 levels:
• eukaryotic message has lots of factors that regulate how efficiently it is translated e.g. secondary structure in 5’ untranslated region
• Global protein synthesis regulation

• This is because cells are really sensitive to the environment, they are growing–>
• Inhibited by cell stresses and the withdrawal of nutrients over a certain period of time: -serum deprivation, temperature shock, DNA damage, viral infection, hypoxia, cytokine treatment= cells start switch down protein synthesis= programmed cell death.

Question 13

• Translation must go fast enough to supply protein but slow enough to avoid too many errors
• Error rate 1 in 104 incorrect amino acid
• Ribosomes add 20 amino acids/second to a polypeptide chain e.g: the synthesis of actin 375 amino acids takes 20 seconds
• Protein synthesis is energetically expensive

Answer 13

mad

Question 14

describe the 80s ribosome

Answer 14

• Not static; in a process of dissociation of its 2 subunits (60S and 40S) and reassociation
• When the ribosome is dissociated into its 2 subunits= it has the most potency to initiate protein synthesis= protein synthesis starts with an alone subunit.
• Small and large subunits bind together during initiation
• Translation takes place in the cavity between the two subunits
• Peptidyl transferase activity (peptide bond formation) associated with the large 60S subunit
• When 2 subunits come back together at the start of elongation–> Has three binding sites for tRNA: E, P, A (involved in the movement of the ribosome along the message during translocation).

Question 15

describe the ribosome-polysome cycle

Answer 15

• A polysome is a structure that consists of multiple translating ribosomes attached to a single mRNA to synthesise the same protein.
• This can occur in a eukaryotic mRNA
• Ribosomes further downstream= have longer polypeptide chains emerging- can be seen by electron micrograph.
• POLYSOME CYCLE Eukaryotic messages are circular= ribosomes don’t have to dissociate- continuously get recycled round and round to initiate protein synthesis.
• This leads to a better insight of the complexity of how mutations, extracellular stimuli, intercellular cues, growth conditions, and stress could lead to the change of translation in the cell

Question 16

where can proteins be obtained from?

Answer 16

For diagnostic purposes, they may be obtained from:
• Patient cells or tissues
• Microorganisms
• Cell lines derived from insects, vertebrate animals, or plants – in vitro cell culture to extract proteins

Question 17

describe the lysis of cell pellets to extract proteins from cells

Answer 17

• detergents are used for the lysis of cell pellets.
• Examples of detergents: Triton X100, NP40, Tween 20 and SDS
• The strength of the detergent dictates which compartment we are breaking e.g. SDS breaks nuclear membrane and the outer compartment of cell (thus whole cell extract) whereas Triton only breaks the outer membrane, keeping nucleus intact= can spin down nuclei take off cytoplasmic extract lyse nuclei with SDS and obtain nuclei extract.
• Can look at differences between proteins in cytoplasm Vs nucleus
• Depending on the protein of interest, proteins can be purified and analysed from a number of extracts:
  • Whole cell
  • Cytoplasmic
  • Nuclear
• Gentle detergent –cytoplasmic extract + nuclear pellet
• Increased detergent strength- whole cell extract

Question 18

Quantification of Protein Extracts
- Once you have made extract= important to quantitate amount of protein in extract e.g. by Bradford assay–>
- Red dye colour, when mixed with protein, changes to blue colour (in the right conditions).
- Work out concentration of protein by using standard curve; get curve by reading a plate using a plate reader.
Once you have quantitated the protein–>

Answer 18

SDS-Polyacrylamide Gel Electrophoresis:

• Separate all the proteins in mixture using SDS polyacrylamide gel electrophoresis
• Discontinuous gel system: separating gel (pH 8.8) + stacking gel (pH: 6.8)
• Running buffer (tris-glycine AA) allows the separation of proteins; only has H as its sidechain= load samples into wells= glycine behaves differently at different pH e.g. protonated or not
• Samples in top gel: will stack/line up–> enter separating gel glycine migrates it at different rate–> separates protein based on their MW.

Question 19

How do you run a SDS-PAGE gel?

Answer 19

• Combine the extract with a sample buffer (as extract is often colourless- would not see it otherwise) = sample buffer contains –>
  1) bromophenol blue and contains glycerol= extract looks blue and glycerol gives it some weight + ensures the extract sinks to the bottom of the well AND STAYS THERE
  2) Tris- hcl= buffer
  3) Distilled Water= makes up for volume= all samples are the same
  4) Beta mercaptoethanol
  5) SDS

Question 20

what are the components of the sample buffer?

Answer 20

• Aim: to break down structures of proteins before loaded onto gel
• SDS breaks noncovalent bonds and adds negative charge to amino acid side chains- correct charge to move to positive anode
• Beta-mercaptoethanol: breaks the disulphide bonds

Question 21

What can we do with gels?

Answer 21

• Stop the gel when it is at the right distance (Dictated by rainbow marker= sample that is loaded on the gel, that can be seen whilst the gel is running. As you know the MW of protein of interest= stop the run when the dye front is at the right position).
• When the gel stops running; can do 2 things—>

1) Take gel apart from the plate= stain the gel using Coomassie blue (Stains protein in every part of extract blue) OR
2) Semi-dry blotting: Take the gel= transfer protein onto a membrane

Question 22

describe semi dry blotting

Answer 22

• Allows the transfer of proteins from the gel to a membrane
• This is achieved by using a blotter 2 plates, 2 electrodes= create a sandwich between them made up of pre-wetted filter paper (soaked in transfer buffer)= stack membrane= put gel on top of that= put more filter paper on top= clamp two parts together= apply electrical current.
• This allows the protein in gel to migrate and attach itself onto the membrane
• Can use membrane in western blotting

Question 23

describe western blotting

Answer 23

• Ability to identify individual proteins within cell extract by using a primary antibody for protein of interest–> recognise antibody using a secondary antibody attached to enzyme–> introduce substrate–> enzyme changes substrate to a specific colour OR (in HRP case)= chemical reaction–> light emission (chemiluminescence) captured on X-ray film containing iamge of the bands.
• Horseradish peroxidase (HRP) conjugated secondary antibodies
• Used in conjunction with specific chemiluminescent substrates (LumiGLO) will generate a light signal.
• In the presence of hydrogen peroxide (substrate), horseradish peroxidase (HRP ) converts luminol to an excited intermediate dianion. This dianion emits light on return to its ground state.
• Light emission is maximal immediately after exposure of the substrate to HRP and continues for 0.5-1 hour. Light can be captured on X-ray film, typically by exposure for a few seconds/minutes

Question 24

RECAP:
Cell culture allows us to grow cell in vitro to extract proteins–> proteins can be quantified–> can separate them using SDS polyacrylamide gel electrophoresis–> can also do blotting= leads to bands of interest

Answer 24

Main difference between semi dry blot and stain gel: stain gels pick up all the proteins in the sample (non-specific) whereas western blot identifies specific proteins within the extract. Western blot also used to detect how well loading is by using antibodies to housekeep genes.

Question 25

What can we identify with stained gels versus blots?

Answer 25

Stained gels:

• Can stain all proteins within extract
• Can also tell you if samples are equally loaded
• Cannot identify specific proteins

Blot:

• Can identify specific proteins with an extract
• Can also probe to look for loading
• Semi-quantitative, can use software to quantitate bands