western blotting.pdf

8/14/2019 Western Blotting.pdf

1/17

1

Western blotting

Western Blot Definition:

Western blotting is a technique used to identify and locate proteins based on their ability to

bind to specific antibodies.

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).

Summary:Western Blot Gives You Information on the:

Size of your Protein

Expression Amount of your Protein

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!


2/17

2

Figure 1. Details of the steps involved in obtaining protein for western blot.

Figure 2. Figure detailing the steps in conducting a western blot.


3/17

3

How Does Western Blotting Work?

See Diagram 1below. 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.

What You Need to Western Blot:

A Protein Sample

A Good Antibody to Detect your Protein of Interest

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 differentproteins!). 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 2shows a western blot example gel. Lane 1 is a protein size marker ladder which

shows different knownsizes 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!

To Detect your Protein:

Buy an Antibody Against Your Primary Antibody Source

Use an ECL - Chemiluminescence Kit and Film to Get the Results


4/17

4

Diagram 1. Diagram 2.

Now Learn everything else there is about western blots!

Steps in Western Blot Analysis:- Sample Prepartation

- Lysing Buffer

- Antibody

- Lysing Cells


5/17


6/17

6

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 Antibodiesare 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.)


7/17

7

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 proteinaggregation ).

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


8/17

8

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


9/17

9

- 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 blackfilter paper

gel nitrocellulose

filter paper

sponge

(+)

Transfer :


10/17

10

- Keep cool in 4C

- Transfer overnight at 35 V

- Can transfer quickly in 1 hour at higherV

Western Blot

Blocking of Membranesallows 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.

- Buffer is TBST

Washing After Blocking

- washing after blocking steps is not critical, as people often block during primary antibody

incubations.

- washing is usually done with TBST buffer. Can be done quickly with a large volume of

TBST.

Incubation with Primary Antibody

- mouse, rabbit or other species

- usually 1 : 1000 dilution with TBST

- if high background/many non-specific bands is a problem, incubations can be done with 5%

BSA or Milk.

Washing after Primary Antibody

- not so critical

- can wash quickly with large volumes of TBST

Incubation of Secondary Antibody


11/17

11

- usually diluted 1 : 10, 000 with TBST

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

Washing After Secondary Antibody

- CRITICAL

- Wash 5 X for 5 minutes with TBST.

- If you really must rush, wash at least 3 times with larger volumes of TBST.

Assaying for Results

- usually ECL (Enhanced Chemiluminescence) is used

- film is exposed and developed. Exposure for 10 seconds is usually enough for total protein.

Phospho-proteins can require 2 - 20 minute exposures.

- 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


12/17

12

Ig - Immunoglobulin(an antibody isotype)

NP-40 - Nonidet P-40

PMSF - phenylmethylsulfonyl fluoride

BSA - Bovine Serum Albumin

NFDM - Non-fat dry Milk


13/17


14/17

14

Table 1. Comparison of direct and indirect detection methods

Direct Detection

Advantages

Quick methodology since only one antibody is used.

Cross-reactivity of secondary antibody is eliminated.

Double staining is easily achieved using different labels on primary antibodies fromthe same host.

Disadvantages

Immunoreactivity of the primary antibody may be reduced as a result of labeling.

Labeling of every primary antibody is time consuming and expensive.

No flexibility in choice of primary antibody label from one experiment to another.

Little signal amplification.

Indirect Method

Advantages

Sensitivity is increased because each primary antibody contains several epitopesthat can be bound by the labeled secondary antibody, allowing for signalamplification.

A wide variety of labeled secondary antibodies are available commercially.

Since many primary antibodies can be made in one species and the same labeled

secondary antibody can be used for detection, it is versatile. Immunoreactivity of the primary antibody is not affected by labeling.

Different visualization markers can be used with the same primary antibody.

Disadvantages

Cross-reactivity may occur with the secondary antibody, resulting in nonspecificstaining.

An extra incubation step is required in the procedure.

Protein Transfer to a MembraneFollowing electrophoresis, the protein must be transferred from the electrophoresis gel to a membrane. Thereare a variety of methods that have been used for this process, including diffusion transfer, capillary transfer,heat-accelerated convectional transfer, vacuum blotting transfer and electroelution. The transfer method that isused most commonly used for proteins is electroelution or electrophoretic transfer, because of its speed andtransfer efficiency. This method uses the electrophoretic mobility of proteins to transfer them from the gel to thematrix. Electrophoretic transfer of proteins involves placing a protein-containing polyacrylamide gel in directcontact with a piece of nitrocellulose or other suitable, protein-binding support and "sandwiching" this betweentwo electrodes submerged in a conducting solution (Figure 2). When an electric field is applied, the proteinsmove out of the polyacrylamide gel and onto the surface of the membrane, where the proteins become tightlyattached. The resulting membrane is a copy of the protein pattern that was found in the polyacrylamide gel itself.


15/17

15

Figure 2. Electrophoretic transfer.

Transfer efficiency can vary dramatically among proteins, based upon the ability of a protein to migrate out of

the gel and its propensity to bind to the membrane under a particular set of conditions. The efficiency of transferdepends on factors such as the composition of the gel, complete contact of the gel with the membrane, the

position of the electrodes, the transfer time, size and composition of proteins, field strength and the presence ofdetergents. Optimal transfer of proteins is generally obtained in low ionic strength buffers and with low electrical

current.

Pierce offers a wide selection of the most commonly used membranes for Western blotting, includingnitrocellulose, polyvinylidene difluoride (PVDF) and nylon membranes.

After transfer and before proceeding with the Western blot, it is often desirable to stain all proteins on themembrane with a reversible stain to check the transfer efficiency. Although the gel may be stained to determinethat protein left the gel, this does not ensure efficient binding of protein on the membrane. Ponceau S stain is

the most widely used reagent for staining proteins on a membrane. However, it has limited sensitivity, does not

photograph well, and fades with time. MemCode Reversible Stain is a superior alternative for staining proteinon nitrocellulose or PVDF membranes. MemCode Stain detects low nanogram levels of protein, is easilyphotographed, does not fade with time and takes less than 30 minutes to stain, photograph and erase.

Blocking Nonspecific Binding Sites

In a Western blot, it is important to block the unreacted sites on the membrane to reduce the amount ofnonspecific binding of proteins during subsequent steps in the assay. A variety of blocking buffers ranging frommilk or normal serum to highly purified proteins have been used to block unreacted sites on a membrane. The

blocking buffer should improve the sensitivity of the assay by reducing background interference. Individualblocking buffers are not compatible with every system. For this reason, a variety of blockers in both Tris buffered

saline (TBS) and phosphate buffered saline (PBS) are available. The proper choice of blocker for a given blotdepends on the antigen itself and on the type of enzyme conjugate to be used. For example, with applicationsusing an alkaline phosphatase conjugate, a blocking buffer in TBS should be selected, because PBS interferes

with alkaline phosphatase. The ideal blocking buffer will bind to all potential sites of nonspecific interaction,eliminating background altogether without altering or obscuring the epitope for antibody binding.

For true optimization of the blocking step for a particular immunoassay, empirical testing is essential. Manyfactors, including various protein:protein interactions unique to a given set of immunoassay reagents, can

influence nonspecific binding. The most important parameter when selecting a blocker is the signal:noise ratio,measured as the signal obtained with a sample containing the target analyte, as compared to that obtained witha sample without the target analyte. Using inadequate amounts of blocker will result in excessive background

staining and a reduced signal:noise ratio. Using excessive concentrations of blocker may mask antibody:antigeninteractions or inhibit the marker enzyme, again causing a reduction of the signal:noise ratio. When developing


16/17

16

any new immunoassay, it is important to test several different blockers for the highest signal:noise ratio in theassay. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique

characteristics.

Pierce offers a complete line of blocking buffers for Western blotting including BLOTTO, Casein, BSA, SEABLOCK and the exclusive SuperBlock Blocking Buffers.

Blocker BLOTTO is a ready-to-use blocking buffer made from nonfat dry milk. Nonfat dry milk containsendogenous biotin and should not be used with avidin-biotin systems.

Blocker Casein is a 1% (w/v) ready-to-use solution of Hammersten Grade casein that can be used toblock nonspecific sites. Since casein is a purified protein, it is less likely to cross-react with theantibodies and cause high background than BLOTTO or other complex protein mixtures.

Blocker BSA is a 10% solution of high quality bovine serum albumin (BSA). BSA is a commonlyusedblocking agent for all immunoassay applications. Blocker BSA is concentrated and must be diluted priorto use. One to three percent BSA solutions are commonly used for blocking nonspecific sites.

SEA BLOCK Blocking Buffer is made from steelhead salmon serum. As a non-mammalian proteinblocker the risk of background caused by nonspecific interactions is minimized.

SuperBlock Blocking Buffer is a highly purified non-serum protein solution that is the ideal blockingagent in many assays. The blocking ability of SuperBlock Buffers and their ability to maintain a highsignal:noise ratio is superior to most other formulations. SuperBlock Blocking Buffers also accomplishblocking very quickly, often in as little as 10 minutes.

StartingBlock Blocking Buffer contains no biotin or serum proteins and is compatible with a wide range

of detection systems. StartingBlock Buffer works in less than 15 minutes on Western blots and almostimmediately in ELISA plates, requiring no incubation step.

Washing

Like other immunoassay procedures, Western blotting consists of a series of incubations with differentimmunochemical reagents separated by wash steps. Washing steps are necessary to remove unboundreagents and reduce background, thereby increasing the signal:noise ratio. Insufficient washing will allow highbackground, while excessive washing may result in decreased sensitivity caused by elution of the antibodyand/or antigen from the blot. As with other steps in performing a Western blot, a variety of buffers may be used.Occasionally, washing is performed in a physiological buffer such as Tris buffered saline (TBS) or phosphatebuffered saline (PBS) without any additives. More commonly, a detergent such as 0.05% Tween 20 (Product #28320) is added to the buffer to help remove nonspecifically bound material. Another common technique is touse a dilute solution of the blocking buffer along with some added detergent. Including the blocking agent andadding a detergent in wash buffers helps to minimize background in the assay. For best results, only high-puritydetergents, such as Surfact-Amps Detergents for Western blotting.(back to top)

Primary and Secondary Antibodies

The choice of a primary antibody for a Western blot will depend on the antigen to be detected and whatantibodies are available to that antigen. A huge number of primary antibodies are available commercially andcan be identified quickly by searching sites such as www.antibodyresource.comor www.sciquest.comonthe internet. Alternatively, a primary antibody may be made to recognize the antigen of interest. For more

information on producing a custom antibody, see the Antibody Production and Purification technical section ofthe catalog. Both polyclonal and monoclonal antibodies work well for Western blotting. Polyclonal antibodies areless expensive and less time-consuming to produce and they often have a high affinity for the antigen.Monoclonal antibodies are valued for their specificity, purity and consistency that result in lower background.Crude antibody preparations such as serum or ascites fluid are sometimes used for Western blotting, but theimpurities present may increase background. To obtain antibodies with the greatest specificity, they can beaffinity purified using the immobilized antigen. For more information on affinity purification, see the AntibodyProduction and Purification technical section of the catalog.

A wide variety of labeled, secondary antibodies can be used for Western blot detection. The choice ofsecondary antibody depends upon the species of animal in which the primary antibody was raised (the hostspecies). For example, if the primary antibody is a mouse monoclonal antibody then the secondary antibody


17/17

17

must be an anti-mouse antibody obtained from a host other than the mouse. The host species of the secondaryantibody often will not affect the experiment. However, secondary antibodies are available from several differenthost species and if a secondary antibody causes high background in a particular assay, another host speciesmay be chosen. Another option to reduce background is to use a secondary antibody that has been pre-adsorbed to serum proteins from other species. This pre-adsorption process removes antibodies that have thepotential to cross-react with serum proteins, including antibodies, from those species. To expedite the processof choosing the appropriate secondary antibody, visit the Secondary Antibody Selection Guideon our website.

Antibodies for Western blotting are typically used as dilute solutions, ranging from 1/100-1/500,000 dilutionsbeginning from a 1 mg/ml stock solution. The optimal dilution of a given antibody with a particular detectionsystem must be determined experimentally. More sensitive detection systems require that less antibody beused, which can result in substantial savings on antibody costs and allow a limited supply of antibody to bestretched out over more experiments. It also produces a side benefit of reduced background because the limitedamount of antibody shows increased specificity for the target with the highest affinity. Antibody dilutions aretypically made in the wash buffer containing a blocking agent. The presence of a small amount of blocking agentand detergent in the antibody diluent often helps to minimize background.

Pierce offers a wide variety of ImmunoPure Labeled Secondary Antibodies for use in Western blotting. Thelabels include biotin, fluorescein, rhodamine, horseradish peroxidase and alkaline phosphatase. For thecomplete list of labeled secondary antibodies,

western blotting.pdf

Documents