biochem lab 9

watch video on western blotting & calibration curve for SDS page & then outline everything

Western blotting is a ___________ technique used to detect a specific __________ in a sample

sensitive, protein

In the last lab, we ________ our proteins according to ________ using _____-___________ and then ___________ the proteins in the gel onto a ___________ membrane

In lab 9, we will _______ the particular _________ on the ________

separated, size, SDS-PAGE, electroblotted, PVDF

Detect, protein, membrane

Today, we will detect our protein of interest using a series of steps that involves an _______ that specifically recognizes and binds to the specific _______

antibody, protein

Today, we will do a second test to see if the transfer of _______ from SDS-PAGE gel to the _________ was successful by staining the proteins on the membrane with the _______ dye Ponceau S

proteins

membrane

red

Unlike ________ ________ ________ , ____ _______ will not interfere with subsequent steps in western blot detection

Coomassie brilliant blue
Ponceau S

The reason we do not use Ponceau S normally after SDS-PAGE is because it is ___ fold less sensitive than Coomassie

Bands that contain less than ________ ng protein will be very faint or not show up at all.

After staining we will take a picture and then wash off the ____ dye and proceed with detection of the His-6 tag

10

200

red

Prior to treating the membrane with an antibody, we must first “_______” the nonspecific protein binding sites remaining on the membrane

After transfer, the membrane still has a high capacity to bind many more proteins → we want the antibody to only bind to ___-____

Blocking solutions contain high amounts of _______ which bind and saturate all the remaining sites on the membrane so that the antibody cannot stick nonspecifically

block

His6-tag

protein

Our goal is to visualize only those proteins on the blotted _________ membrane that contains a _____________

After the blocking step a primary _________ that recognizes the his6-tag is added to the membrane —> an ___________ __________ primary antibody has been chosen (incubation with a ___________ antibody is not required) → we can directly detect our primary antibody

PVDF

His6tag

antibody

enzyme conjugated

secondary

Our anti-His6 primary antibody is covalently attached to the enzyme _________ _______ (AP) → AP is an enzyme that _________ a large variety of compounds → AP will hydrolyze the substrate 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)

An intermediate formed dimerizes to form an indigo purple product in the presence of the chromogen Nitroblue tetrazolium (NBBT) → Thus we will see a purple band, visible to the eye, on the membrane wherever there is a his6 tagged protein

alkaline phosphatase

dephosphorylates

To quantify the protein of interest a method is used that relies on the antibody being conjugated to a near ______ _______ ________ instead of being conjugated to an enzyme such as alkaline phosphatase

Because the dye is not an enzyme, its presence can be directly detected without addition of a __________. The IRDye 800CW is excited at ______ nm, and its emision is detected at near _____ nm.

Other disadvantages of IRdyes are that they are 1) very sensitive 2) Faster as no substrate is required and 3) stable and the blot can be re-imaged months later

Example of a modern IRdye detector is LI-COR Odyssey Fc, which can detect the signal through a very large dynamic range. The signal can then be quantified using special software to determine how the levels of ________ differ in each lane

Infrared flourescent dye

Substrate

774, 800

Protein

Reading a calibration curve

1. C1V1=C2V2 for 6X bradford

2. C2V2=C2V2 for BSA

3. subtract to get water

1. solve for x

2. concentration of storage vial: divide big by small dilution and multiply b X

3. total yield: mulitply concentration x total volume→ convert to micromolar by ug/mL x mL/L x 1 mmol/MW of protein (uM)

Several factors must be considered when setting up a western blot

1) size, type and abundance of the protein of interest

2) type of membrane used for transfer

3) availabiity of the primary antibody (Ab)

4) choice of the covalently linked enzyme and detection system

In lab 8 the MW will determine the correct _______ ________ ______

% polyacrylamide gel (high mw = low %)

In sds pgae, due to the presence of the detergent sds, all proteins become _______ and are _______ charged and migrate toward the (cathode/anode)

A sandwich is made with

Gel is closer to (cathode/anode) and membrane is closer to (cathode/anode)

An electric current is applied and the proteins move out of the _______ and onto the ______

Denatured, megatively, anode (+)

Fiber pads, filter paper, sds page gel, membrane

cathode, anode

Gel, membrane

The primary Ab will bind to the _______ of ______ on the membrane, the _______-________ site of the primary ab _______ and ______ tightly to a particular part of the protien of interest called the _______

The secondary Ab will bind to the _________ region (constant region) of the primary Ab or stem part of the y shaped protein. Secondary antibodies are covalently linked to an enzyme that can catalyze the conversion of a ________ to a detectable ________. The product is usually chemiluminescent or colored.

Two enzymes that are commonly covalently linked to antibodies for immunodetection are _________ ______ and _______ ________

1. Is an enzyme that can convert a particular substrate to produce _________ (chemiluminescence)

2. Is an enzyme that ___________ a large variety of compounds —→ its activity can be detected by adding a phosphate substrate called ________ and the chromagen _________ ________ → these two react to form a _____-_____ precipitate

Negative control is and positive control is ______. If western blot worked correctly we should see a _____-______ band in positive control band

Protein, interest, antigen binding,recognizes, binds, epitope, Fc, substrate, product, horseradish peroxidase, alkaline phosphatase, light, dephosphorylates, BCIP, nitroblue tetrazolium, bluish purple, e coli cell with no his 6 tagged protein, udgx that was purified previously, blue purple