Western Blotting

what does western blotting quantify

proteins

What are the 5 steps of western blotting

1. Extract the total protein

2. Run a Bradford assay on BSA and the samples 3x. Then take the average and create a standard curve and find the concentration of the samples.

3. Dilute the samples accordingly. Heat them and mix them with SDS and Bmercaptoethanol. Then load the same amount of protein into each well of the SDS-PAGE gels. Do this 3x

4. Blotting/transfer step

5. Add antibodies to your iblot membrane depending on the chosen method

Where do you extract the total protein sample from

A tissue or cell

what can you do if you extract the protein and it’s not highly expressed

concentrate it

what does BSA stand for

Bovine serum albumin

What kind of dilution do you run on the BSA for your Bradford assay

Serial dilutions

Describe the Bradford assay step for BSA

(Draw it)

1. Get a known [BSA] and do serial dilutions 3x

2. Take the ABS of each

3. Make a standard curve using the average

Describe the Bradford assay step for your samples

(Draw it)

1. Dilute your samples if needed so that they would likely fall in the linear range

2. Run the assay 3x

3. Take the ABS of each

4. Take the average ABS

5. Plug in the ABS into the linear equation to get the concentration

What went wrong if one of the ABS sample values is 1.3

You didn’t dilute the sample enough

What went wrong if one of your BSA ABS values for 0.3 is 1?

You made a mistake in your serial dilutions. Could be a miscalculation or you didn’t dilute it enough.

If the linear equation is y=.25x+.2

And your ABS sample values are

S1 = 0.72

S2 = 0.5

S3 = 0.36

What are their concentrations?

S1 = 2.88

S2 = 2

S3 = 1.44

What does SDS - PAGE gel stand for

Sodium dodecyl sulfate - polyacrylimide gel electrophoresis gel

Using the previous sample concentrations, what 2 things must you do before you’re able to load them into the gel

1. Dilute them all to the same concentration

2. Heat the and mix them with SDS and Bmercaptoethanol

calculate how you must dilute each sample so that there is 5ug of total protein in each well

Why do you heat the total proteins and mix them with SDS and Bmercaptoethanol?

It results in the unfolding and coding of the subunits of the proteins with a negative charge

How many times should you run the protein samples in the gel

3x

What can go wrong during the gel loading step

1. You squirt the protein too fast and it overflows

2. A bubble gets in the gel well and pops, contaminating other wells

3. The current is too high and cooks the proteins

4. You don’t use gloves and contaminate the proteins

What is the order of the gel sandwhich in the blotting/transfer step

(Draw it)

What can go wrong during the blotting/transfer step

1. It’s not rolled out smoothly

2. There’s bubbles in it

draw the antibodies for finding Bactin with chemi-luminescence

draw the antibodies for finding Bactin with IR. What are 3 did IR spectrums you can use? What machine do you need for it?

What is an advantage to using IR over chemi-luminescence

You can stack 2 dif protein band readings on 1 blot.

What reference genes can be used and what’s their size?

GAPDH ~ 35kDa

Bactin ~ 40kDa

How much space do you typically need between gel selection

5-10kDa

What can go wrong while you’re taking pictures of your iblot

1. If you’re running multiple samples that you want to compare, you don’t use the same excitation emission and timed exposure

2. If you’re doing chemi-luminescence: you don’t let the enzyme sit long enough

3. You don’t run the exposure long enough

CAPA is an inhibitor of protein X. You want to test its affects on protein X (30kDa) expression. Describe how the experimental groups were setup. Describe your experimental groups. Draw your results.

Control: protein X without CAPA, N=3. Sample extracted from mouse that didn’t have CAPA.

Reference gene: Bactin (40kDa), N=3. Sample extracted from mouse that didn’t have CAPA.

Treated group: protein X with CAPA, N=3. Sample was extracted from mouse that was given CAPA via food 3x a day for a week prior to extraction.

Must include: ladder, reference gene, control group, treated group

CAPA is an inhibitor of protein X. You want to test its high vs low dosage affects on protein B (45kDa) expression. Describe how the experimental groups were setup. Describe your experimental groups. Draw your results.

Negative Control: protein B without CAPA, N=3. Sample extracted from mouse that didn’t have CAPA.

Reference gene: GAPDH (35kDa), N=3. Sample extracted from mouse that didn’t have CAPA.

Positive Control: protein B with CAPA, N=3. Sample was extracted from mouse that was given 10ug CAPA via food 3x a day for a week prior to extraction.

Low dose group: protein B with CAPA, N=3. Sample was extracted from mouse that was given 5ug CAPA via food 3x a day for a week prior to extraction.

High dose group: protein B with CAPA, N=3. Sample was extracted from mouse that was given 20ug CAPA via food 3x a day for a week prior to extraction.

Must include: ladder, reference gene, positive control group, negative control group, high dose, low dose