Immunology Lab Final
Describe the Western blot procedure?
1. Pour Gel - Run SDS Gel - separates protein in polyacrylamide matrix 2. Transfer to Nitrocellulose -proteins removed from gel and now on membrane 3. Run in electric current - - Negative proteins travel toward positive cathode 4. AFTER TRANSFER -remove blot (nitrocellulose) from transfer cassette 5. Block-using Milk protein to bind all spots on membrane where no proteins are found 6. Primary Antibody Incubation-Using - mouse antiHIV IgG 7. Washing- to remove excess 8. Secondary Antibody Incubation - using goat anti mouse IgG 9. Washing- to remove excess 10. Substrate - (ECL plus) for production of light and detection of protein 11. Detection- Chemilluminescence imager
What Alexa is being used in the immunofluorescence lab?
488
What are the two common Alexa Fluorochromes? What do they emit at and what color is produced?
Alexa 488 (emits at 519-green) and Alexa 568 (emits at 630-red)
What are applications of IF?
Clinically it can be used to identify the presence of apathogen in a patient sample
What did we test for in the ELISA?
Looking for presence of HIV Ag in patient sample to determine which of two treatments are better.
What are applications of the Dot Blot?
Routinely used in research and clinical labs • Can test for the presence of an antibody in a patientsample to determine exposure to a pathogen • Can test for the presence of an antigen in a patientsample to determine if infected
What is Excitation?
The movement of an electron to a higher energy level
Compare between Sandwich ELISA and Non-Competitive (Indirect) ELISA
The sandwich ELISA is used for sensitivity and specificity. This ELISA also begins with the antibody. The non-competitive ELIZA is the indirect method that begins with the antigen sample.
What is the purpose of the block in the dot blot?
add nonspecific protein to bind to all spots on nitrocellulose that the protein of interest didn't bind to
What is the purpose for the 20% glycerol portion in the loading buffer of the western blot experiment?
adds weight to the sample so it sinks to the bottom of the well
What is the purpose of the secondary antibody in the dot blot?
binds specifically to primary antibody. Hasan enzyme conjugated to it.
What is in the sample (loading) buffer? (6)
-2% SDS -20% glycerol -20 mM Tris-Cl, pH 8 -2 mM ethylene diamine tetraacetic acid (EDTA) -dithiothreitol (DTT) or 2-mercaptoethanol -bromophenol blue
Describe the ELISA procedure
1. Label the outside of one of the two 12-well strips 1-12 and the other labeled 3 positive, 3 negative, three A and 3 B 2. Use a pipet to add 50μL of PBS sample to each of the wells #2-12. DO NOT add this sample to well #1. 3. Add 100μL from the sample labeled 1,000ng/mL AG to well #1 and begin serial dilution. 4. In the 12-well strip, use a fresh pipet tip to transfer 50μL of the positive control to its designated 3 wells (1-3). 5. Change the pipet tip and add 50μL of the negative control to its designated 3 wells (4-6). 6. Change the pipet tip again and add 50μL of sample A and sample B to designated wells 7. Allow this 12-well strip to sit for 5 minutes while the samples bind to the plastic. 8. Fill each well with buffer to wash the wells after tapping. 9. Use a new pipet tip to add 50μL of the primary antibody solution into all 12 wells and let it sit for 5 minutes to allow for the primary antibody to bind. 10. Once 5 minutes pass by, tip the 12-well strip upside down and tap on a paper towel to rid of solution that is not bound. 11. Using a new pipet tip, add 50μL into all 12 wells of the strip and then wait 5 minutes to let the antibody bind. 12. Wash 13. Add 50μL of the enzyme substrate into all 12 wells and wait 5 minutes (DO NOT RINSE). 14. After waiting 5 minutes to allow the enzyme substrate to bind, add 50μL of 0.18M sulfuric acid to each of the two 12 well strips and read them at 450nm with a spectrophotometer.
Describe the transfer process in the Western Blot.
1. Staring with the BLACK side of the transfer apparatus place an electro-blotter pad down, then filter paper, and gel, on top of gel lay PVDF (Nitrocellulose), then filter paper, and electro-blotter pad on the RED/Clear side of the apparatus. 2. The red side is the positive cathode, which is the direction the negatively charged proteins will travel when under electric current. We want the PVDF (nitrocellulose) tocatch the proteins as they run out of the gel towards the positive cathode. Therefore it is extremely important for your PVDF (nitrocellulose) to be on the red side and your gel to be on the black side of the transfer apparatus. 3. Place the sandwich in the transfer apparatus using the rails on the side and run the transfer at 250 milliAmps for 1 hour. 4. When the blot is done transferring disassemble the sandwich. Throw the filter paper andempty gel in autoclave bucket. SAVE the thick electroblotter pads. Put the PVDF(protein side up) into the milk block in step 3.
How are the antibodies made in the dot blot experiment?
Antibodies are made by infecting animals with an antigen and forcing them to produce antibodies to that specific antigen.
What is the purpose of the primary antibody in the dot blot?
Antibody that will bind to the protein of interestin our sample
What was the block? Why is it used in immunofluorescence lab?
BSA (bovine serum albumin)/MILK to insure that allsurfaces are covered either by cells or an inert protein like BSA
Why do we need controls?
Control samples are necessary to be sure your ELISA is working correctly and to rule out any false negative/positive results.
What is in the Mounting media and why is it important in immunofluorescence?
DAPI which is used as a way to locate and visualize the nucleus of the cells.
What is the meaning of the percentages of the two gel layers in the western blot experiment?
Different % of polyacrylamide can give you different pore sizes for proteins to run through.
Differentiate between direct and Indirect Immunofluorescence?
Direct fluorescence, it is more common and improves the visualization - because multiple second antibodies can bind to the primary antibody, leading to an increased signal from the indicator compared to direct fluorescence). Indirect fluorescence is different because it uses a single secondary antibody directed against the primary antibody.
What is the purpose for the 2% SDS portion in the loading buffer of the western blot experiment?
Gives the sample a negative charge
What were the Secondary Antibodies used?
Goat anti-mouse IgG is used as the secondary antibody
What did we test for in the Western blot?
HIV antibody (positive exposure)
What does rabbit anti-Goat IgG mean?
IgG antibodies made by a rabbit that are produced from infecting it with goat viral antigens
What is the transfer membrane and why can't it be touched without gloves?
Polyacrylamide is the transfer membrane and is a neurotoxin and touching without gloves can cause false positive results because of possible cross-contamination with proteins found in the skin of bare hands.
What gives all proteins in the sample of a western blot experiment a negative charge in a wide pH range?
Sodium dodecyl sulfate
What were we testing for in the Dot Blot?
Use the DOT BLOT to determine if a patient is infected with thepathogen Hepatitis B, by detecting the Hepatitis B surface antigen (HBsAg) in patient sample
What are the applications of the Western Blot procedure?
Used to identify/quantify a SPECIFIC protein in a sample using an antibody.
What was the Block? Why is it needed?
We block with Milk proteins from dried carnation milk The reason for the block is nitrocellulose needs to be blocked at all spots that have not received protein during the transfer (to prevent incorrect binding).
What is emission?
When an electron falls to a lower energy level, a photon is emitted
Explain false negatives
a false negative is a test result that indicated that a person does not have a virus/condition when they actually do have it.
Explain False Positives
a false positive is a test result that indicates that a person has a virus/condition when they actually do not have it
What is a Fluorchrome?
a molecule that has the ability to absorb invisible, ultraviolet radiation, and then emits or releases the absorbed energy in a manner that can be seen with the human eye. A compound that absorbs light at onewavelength and emit light at a second wavelength
What are the two layers of gel used in the Western blot experiment? What are the percentages of the layers?
a stacking layer (4%) and resolving layer (10%)
What was the Enzyme-Substrate system used for detection?
chemilluminscence imager
Describe the theory of the ELISA experiment
detection of antibodies/antigens in a sample.
What were the secondary antibodies used and flourochromes attached in your immunofluorescence experiment?
goat-anti mouse IgG Alexa 488
What is the purpose for the 20 mM Tris-Cl, pH 8 portion in the loading buffer of the western blot experiment?
maintains the negative charge
How does the fluorescent Microscope aid in the excitation/emission process in immunofluorescence?
maximum excitation takes place at 488nm and maximum emission takes place at 540nm (we are reading at 488 so we are reaching maximum excitation)
What is the purpose for the 2 mM ethylene diamine tetraacetic acid (EDTA) portion in the loading buffer of the western blot experiment?
minimizes protein degradation
What were the Primary Antibodies used in the western blot and what proteins do they bind?
mouse anti-HIV IgG is the primary antibody being used and it binds to the protein of interest (HIV) on the nitrocellulose
What were the cells used in the immunofluorescence lab? What should we expect to see?
nasal epithelial cells; if COVID-19 is present, then there will be a fluorescent signal of Alexa 488
Is there a substrate used in immunofluorescence?
no
What is the name of the matrix used in the western blot?
polyacrylamide matrix
What is the purpose of the substrate in the dot blot?
reacts with enzyme to produce a detectable product
What is the purpose for the dithiothreitol (DTT)/2-mercaptoethanol portion in the loading buffer of the western blot experiment?
reducing agent, breaks the sulfide bonds and linearizes the protein
What is the purpose of the wash in the dot blot?
rid of any solution that was added that did not bind irreversilby
How are the proteins separated in the matrix of the western blot? What is this based on?
separated using electricity to stick to nitrocellulose; this is based on the size of the protein
What does SDS-PAGE stand for?
sodium dodecyl sulfate polyacrylamide gel electrophoresis
What is the purpose for the bromophenol blue portion in the loading buffer of the western blot experiment?
visualization of the sample
What are Clinical/Industrial Applications of ELISA?
Detect the presence of an Antibody: • HIV infection • RSV infection Quantify the Antigen/Antibody in a sample.
What are Home Applications?
Detect the presence of an Antigen • Drug Test • Pregnancy Test
Describe the theory of the Western blot experiment.
Detection of specific proteins in a sample
What was the purpose of the immunofluorescence lab?
Determination of the presence of the COVID-19 pathogen in a patients sample.