Western Blot

Written by Super User. Posted in Protein

Learn about the Western Blot technique.

 

Western Blot - Western Blotting

   Western blot analysis can detect your protein of interest from a mixture of a great number of proteins.  Western blotting can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another cell type or tissue). Western blot analysis can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro. Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how specific it is for this protein. Antibodies are now easily obtainable from commercial sources, and you can purchase one for your protein of interest. If your protein is a novel protein, you must produce an antibody yourself or get a company to do it for you. In this case you will need at least a small amount of your protein either purified from cell extracts or made as a recombinant (ie in vitro or in a recombinant protein expression system).   Antibodies specific to your protein are vital to western blotting as they are able to bind specifically to your protein of interest instead of the thousands of proteins on your western blot!

How Does Western Blotting Work?

See Diagram 1 below. First things first. Obtain a protein sample you want to analyze, such as cell samples. Lyse the cells to release protein contents. Run these on a gel which separates proteins on the basis of size. Then transfer these gel proteins onto a membrane using electricity. This membrane can then be used to probe for proteins of interest using a primary antibody. Western blot relies on the primary antibody to detect this protein from the thousands of proteins on your membrane and previously on your gel! (a cell can contain 30,000 different proteins - and these same proteins can even be altered giving you over 300,000 different proteins!). Using an antibody recognizes your primary antibody (a secondary antibody) you build up a protein-antibody-antibody sandwich!  The secondary antibody has a horse radish peroxidase enzyme which converts a luminol substrate to a light releasing substance! This light is detected as a spot on film. From this spot you can determine how much protein is there relative to other spots, or the size of the protein relative to a size marker that is run also on the gel. Diagram 2 shows a western blot example gel. Lane 1 is a protein size marker ladder which shows different known sizes of proteins, this can be purchased commercially and the sizes of all the spots are given in a pamphlet. Lane 3 is a cancer sample and lane 5 is a normal sample. As you can see the protein in lane 3 has a higher expression than the cancer sample in lane 5, which is interesting. Also, the protein spots in lanes 3 and 5 are the same size as the 2nd spot in the size ladder from lane 1. We can then look at the known protein size from our brochure which we received with the ladder. We then determine that the size of the protein is 80 kDa. Our protein of interest is also 80 kDa. So we know that the western blot worked and that the protein is highly expressed in a cancer sample!

Diagram 1.                                                                                                                                      Diagram 2.

westernblot example gel

Learn everything there is about western blots!

Table of Contents

Steps in Western Blotting:

- Preparing for Western Blot

- Which Antibody to use for Western Blots

- Lysing Cells for Western Blot

- SDS-PAGE Gel Information

- SDS-PAGE Gel Preparation for Western Blots

- Gel Electrophoresis - running the gel

- Tranfer of Proteins to Membrane for Western Blotting

- Western Blot

Our Western Blot Protocol Stripping and Re-probing Protocol

External Links to Other Western Blotting Procedures and Methods

Troubleshooting Western Blots

Terms and Definitions used in Western Blot Analysis

Steps in Western Blot Analysis:

- Sample Prepartation

- Lysing Buffer

- Antibody

- Lysing Cells

- Gel Preparation

- Running Gel

- Transfer of Gel

- Controls

- Western Blot

- Readout

- Analysis and Interpretation

To Prepare Samples for Western Blot:

Whether you have Non-adherent Cells (such as T-cell lines or peripheral blood cells) or Adherent cells (such as Fibroblast cells or COS cells), you need to remove extraneous proteins from the media. For non-adherent cells you can gently pellet them with centrifugation and then wash the pellet with PBS. For adherent cells, simple wash the flask or dish with PBS prior to western blot.

Western blot analysis examines proteins, and so we want the proteins to be release from the cells and also prevent the proteins from being cut up by proteases. We need these to western blot:

- to keep things cold! 4C on ice during cell lysis for western blotting

- use Detergent to break up membranes of cells to release proteins

- Buffer

- Inhibitors (both protease inhibitors and phosphatase inhibitors if we will do western blot for phospho-proteins)

Detergent - Lysing Cells For Western Blot

SDS and RIPA are detergents that are used to solubilize proteins for later analysis using western blotting. This is required as many proteins are either inside the cell or located inside cell membranes, so we need to release these proteins to be able to immunoblot for them.



 

Which Antibody should I use for Western Blotting?

You should select an Antibody to use for Western Blot. Usually either mouse monoclonal antibodies or rabbit polyclonal antibodies are used.

You can use either an antibody without any added groups, or use an antibody which is conjugated to biotin. These antibodies do not require

Polyclonal vs Monoclonal Antibodies for Western Blot

Monoclonal Antibodies are usually better for Western Blotting due to:

- better signal to noise ratio ( lower background in western blot )

- their higher specificity ( you get less contaminating proteins or Ig )

- overall cleaner results on the western blotting film (less non-specific bands detected)

Polyclonal Antibodies are better suited for Immunoprecipitation:

- they recognize more epitopes

- have higher avidity

Lysing Cells for Western Blot:

TIPS before you lyse for western blotting:

If you are going to western blot for protein mass (ie a protein that is not phosphorylated):

- you can lyse in larger volumes (keeping the rest to blot for other proteins)

If you are going to western blot a phospho-protein (or phosphorylated protein) it is important to do the following:

- make sure you use phosphatase inhibitors ( such as vanadate because phosphatases will remove the phosphates from your proteins! )

- also lyse cells in the smallest volume possible ( ie lyse in 100 ul loading buffer, this is done because you can load most of the cells you lysed. This is important as signal is quite weak when western blotting for phospho-proteins.)

If you are looking at protein-protein interactions:

- do not use SDS as it dissociates protein-protein interactions and thus proteins are denatured (especially after boiling)

- for western blotting protein-protein interactions, ie native interactions you must use a less-stringent detergent such RIPA.

Lysing:

- can be done directly on the plate or dish

- pellet the cells

- Keep on Ice and cold - use an ice bucket

- Rule of thumb for how much lysis buffer to use is: 10^5 cells / uL of lysis buffer

- collect in eppendorf tubes and label ( keep these on ice )

Measure total protein before Western Blot Analysis:

- this allows more quantitative analysis if you want to compare treatment conditions in western blot analysis

- commercial kits are available such as a Bradford assay for measuring total protein (from Bio-Rad)

- 0.1% of SDS or greater can interfere with protein measurement

Denature Protein Samples :

- add protein loading sample buffer to an aliquot of cell lysate - contains dye (too see the migration on a gel, 2% SDS)

- add disulfide reducing agent such as beta - mercaptoethanol

- Boil in water bath or heating block (lower temperatures such as mid - 90 degrees decrease protein aggregation ).

- Poke a hole in cap of each tube to prevent popping

SDS-PAGE Gel Information for Western Blotting

SDS-PAGE Gels are gel matrices which are used to separate proteins by size in the presence of electric current. Low percentage gels separate larger proteins whereas higher percentage gels separate smaller proteins better. The problem is that all proteins have a charged associated with them, and in an electrical current this could cause problems. This is solved by the addition of SDS to the protein samples. SDS binds to proteins every few amino acids and neutralizes the charge differences that proteins have. This allows proteins to be separated by size and not by charge.

SDS-PAGE Gel Preparation for Western Blot

- depending on how much protein you will want to load for western blotting, you should use small combs or larger combs.

- the percentage of acrylamide is important. Usually 10% acrylamide is used.

- A resolving gel is used at the bottom with a pH of 8.8

- A stacking gel (4-5%) pH 6.8 is used to pack proteins in together after loading

- Polymerization of gels is increased by the catalysts APS and TEMED which speed up the polymerization reaction (formation and solidification) of the poly-acrylamide gel.

- Load every Sample carefully and slowly to prevent sample leaking out of the lane.

- Load every lane and use sample buffer in each (to prevent differences in ).

 

Gel Electrophoresis

  1. Centrifuge samples for 10 sec after boiling.
  2. Load sample into each lane
  3. Load MW reference
  4. Run gel at 100V (constant voltage) or even better at 40 mA (constant current). Watch protein marker/ladder or dye front for when to stop gel. Constant current gives better and sharper results if you have the time.

- watch for bubbles between glass and under the gel - this means that it is working

- watch for protein migration (should be going down) - if not you have switched the leads and can lose all your samples!

- Initially run slowly through the stacking gel (~ 50 Volts), this gives sharper bands.

- Do not run overnight

- Do not over-heat the gel (this causes the gel to lose rigidity, leading to poor resoloving and blurry badly formed bands)

Tranfer of Proteins to Membrane for Western Blotting

Select Membrane Type:

Can use either:

- PVDF membrane for western blots

- Nitrocellulose membrane for western blots

- Nylon membrane (rarely used now - can tear)

Tips for Transfering to Western Membrane:

-Wear Gloves at all times! Use forceps

- Minimize touching/forceps to the membrane

 

Bio-RAD transfer order:

(-)
sponge on black
filter paper
gel nitrocellulose
filter paper
sponge
(+)

Transfer :

- Keep cool in 4C

- Transfer overnight at 35 V

- Can transfer quickly in 1 hour at higher V

Western Blot

Blocking of Membranes allows you to maximize signal:noise ratio

- wear gloves

- block with 5% Blotto (non-fat dry milk - powder) or 1 - 5% BSA (Bovine Serum Albumin) for phosphorylated proteins

Incubation with Primary Antibody

- mouse, rabbit or other species

- usually 1 : 1000 dilution

Incubation of Secondary Antibody

- usually diluted 1 : 10, 000

- anti-mouse, anti-rabbit, or other antibody conjugated with HRP enzyme for detection

Assaying for Results

- usually ECL (Enhanced Chemiluminescence) is used

- film is exposed and developed

- film is usually XOMAT or similar 'fast' film

Terms used in western blotting:

WB - western blot

IB - immunoblot (another term for western blotting)

SDS - Sodium Dodecyl Sulfate (a detergent used in many western blotting solutions)

PAGE - Polyacrilamide Gel Electrophoresis

Ab - Antibody (antibodies are used to detect your protein of interest)

HRP - Horse Radish Peroxidase

Luminol - substrate that HRP cleaves to cause light formation

ECL - Enhanced Chemiluminescence ( western blot solution which enhances light formation from HRP + Luminol)

kD/kDa - kilodalton (most proteins average between 30 - 80 kDa)

RAM - rabbit anti-mouse Ig

Ig - Immunoglobulin(an antibody isotype)

NP-40 - Nonidet P-40

PMSF - phenylmethylsulfonyl fluoride

BSA - Bovine Serum Albumin

NFDM - Non-fat dry Milk