Antibody methods Flashcards

Question

What is ELISPOT?

Answer 1

Used to determine if the cells are producing something e.g. growth factors, antibodies, hormones and cytokines.

Answer 2

Coat your plate with an antibody then block it. Add your cells and incubate them to produce proteins. After incubation, wash your plate to remove the cells. Then add the secondary antibody which is bound to an enzyme. Then add the substrate for the enzyme. It will form precipitates - each cell producing the protein will form a dot.

Answer 3

The wrong antibody may have been used. The reagents may have gone off. Too much or too little signal - use more or fewer cells, or more or less fluid - by titrating the antibody reagents or fluid.

Answer 4

SDS-PAGE and then western blotting. PAGE = polyacrylamide gel electrophoresis.

Answer 5

Get the proteins out of the cell or supernatant Measure how much protein there is. SDS-PAGE - linearise the protein then separate out by weight. Find the protein using Western Blot.

Answer 6

Detergent reacts with membrane, dissolves it, then intracellular components are released. Common detergents are Tween, Triton, NP-40.

Answer 7

Need to use the same amount of protein from each sample. Use Coomassie-blue to react with the proteins, the more brown the more protein there is. Use a spectrophotometer, proteins are measured at 280nm.

Answer 8

The known concentration of protein is diluted so there is a precise amount in solution. Add Coomassie-blue, then record the absorbance at the wavelength to make an absorbance concentration curve. Then can use a precise equation to known exactly how much protein is in the sample so the reagent concentrations are correct for western blotting.

Answer 9

Polyacrylamide gel - a mixture of acrylamide and bis-acrylamide. Altering the ratio between the reagents changes the pore size - high molecular weight proteins are separated at low %gel. The total acrylamide concentration in the gel affects the migration of proteins through the matrix.

Answer 10

Sodium Dodecyl Sulphate polyacrylamide gel electrophoresis. It is a denaturing gel so the proteins are linearised and there is only one molecule.

Answer 11

Measure equal amounts of proteins, in gel. Add denaturing buffer that has SDS, binds to proteins and gives it a negative charge, so it is separated only be molecular weight. The amount of charge depends on how much protein there is. Add 2B-mercaptoethanol to break disulphide bonds. Add glycerol so samples are more dense and sink into the well. Add bromophenol blue so can track the sample, and indicate pH - yellow/brown = acidic so needs neutralising.

Answer 12

Heat it to 95 degrees for 10 minutes, then place on ice. This breaks weak hydrophobic bonds. The sample is now charged, linear proteins.

Answer 13

It is a vertical thin gel with two types. Stacker gel and Resolving gel. There is also a running buffer of Tris: Glycine. Glycine runs slower than the slowest protein.

Answer 14

It has Tris/HCl at pH 6.8. It is large pore acrylamide. The samples are loaded onto, and the proteins are concentrated.

Answer 15

It has Tris/HCl at pH 8.8. It is small pore acrylamide. This separates out the proteins by size.

Answer 16

Tris-HCl to maintain pH. SDS to maintain denaturation. Glycine.

Answer 17

Coomassie-blue stain shows all the proteins, it is a good control. Silver stain can detect low amounts of proteins, but the gel cant be used pass this point. A ladder is required to know how big the proteins are. Doesn’t tell you about the particular protein so then western blot.

Answer 18

After the PAGE: Verifies that the protein has been expressed. Determines the relative amount of a protein. Analyses protein-protein interactions.

Answer 19

Denaturing - SDS-PAGE. Non-denaturing - native PAGE, so the proteins stay in their complexes.

Answer 20

The gels are very flimsy so antibodies can't be put straight onto the gel. Instead proteins are transferred onto a more robust membrane.

Answer 21

The gel is put on a membrane, with filter paper on either side. Filter paper is then sandwiched between the electrodes. The reagents need to be wet so that the proteins can transfer between gels. The proteins are negative, so go the anode, so the membrane needs to be on the correct side of the gel. See picture.

Answer 22

The electric current pulls proteins from the gel into the nitrocellulose of PVDF membrane. Transfer buffer is used, containing Tris, Glycine and methanol. Transfer can then be checked using a non-permanent stain - Ponceau S.

Answer 23

Blocking. Incubation with primary antibody, then washing membrane. Incubation with secondary antibody, then washing membrane. Incubation with enzyme substrate. Exposure to photographic film/ detector.

Answer 24

Block the membrane with non-specific proteins. The proteins bind to unbound regions of the membrane. It can then be incubated with the primary antibody, which will only bind to the specific protein. This avoids false positives.

Answer 25

If the blocking is too high there will be a low signal. If the blocking is too low there will be a high background.

Answer 26

Bovine serum albumin Casein Gelatin Ovalbumin Fat-free milk, which is often used because it is the cheapest, and can be used for lots of proteins so is efficient.

Answer 27

After blocking, the primary antibody is diluted in blocker. Dilution is based on its titre - if titre against target protein is high (good binding) then increase dilution before applying to the membrane. Incubate for 1-2 hours at room temperature.

Answer 28

Washed with PBST, more than once, to get rid of primary antibody. The time and number of washes is antibody dependant. Usually 3 x 15 minutes at room temperature.

Answer 29

Phosphate buffered saline + Tween 20

Answer 30

The secondary antibody is diluted in blocker. Incubation varies, normally 5 minutes to 1 hour. The antibody must bind to the primary e.g. anti-rabbit to bind to a rabbit primary. Secondary antibody is needed to amplify the signal, so the antibody can be visualised.

Answer 31

Many steps where errors can occur. Large amount of sample needed Semi quantitative through comparing bands. Time consuming protocol Cannot give localisation data, need to use immunofluorescence on slides.

Answer 32

1.5g of NaCl per 100ml. For 1000ml need 15g. 2mM Tris, molar mass 121.14g/mol. Mass = molarity x volume x molar mass. 0.002M x 1L x 121.13g/mol = 0.242g. 7g powdered milk per 100ml. 1000ml = 70g milk. 0.02ml of Tween per 100ml. 1000ml = 0.2ml Tween. Dissolve 15g of NaCl, 0.242g Tris, 70g powdered milk in 800ml distilled water, add 0.2ml Tween and mix. Then bring total volume to 1000ml with distilled water.

Answer 33

Incomplete transfer of proteins from the gel to the membrane. Uneven blocking, causing nonspecific binding - background noise. Insufficient washing, leaving residual antibodies. Protein loaded differently. Membrane may have defects or folds.