Western Blot - General Tips/Procedures and Troubleshooting

The Western blot (WB) (alternatively, protein immunoblot) is an extremely useful analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

General Tips/Procedure for Western Blotting

1.Sample lysis

Preparation of lysate from cell culture

a.	Place the cell culture dish in ice and wash the cells with ice-cold PBS.
b.	Aspirate the PBS, then add ice-cold lysis.
c.	Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube.
d.	Maintain constant agitation for 30 min at 4°C.
e.	Spin at 16,000 x g for 20 min in a 4°C pre-cooled centrifuge.
f.	Gently remove the tube from the centrifuge and place on ice. Transfer the supernatant to a fresh tube kept on ice and discard the pellet.

Preparation of lysate from tissues

a.	Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases.
b.	Place the tissue in round-bottom microcentrifuge tubes or Eppendorf tubes and immerse in liquid nitrogen to “snap freeze”. Store samples at -80°C for later use or keep on ice for immediate homogenization. For a ~5 mg piece of tissue, add ~300 μl lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2 x 300 μl lysis buffer, then maintain constant agitation (for example, on an orbital shaker) for 2 hours at 4°C.
c.	Centrifuge at 16,000 x g at 4°C in a microcentrifuge for 20 min. Gently remove the tubes from the centrifuge and place on ice. Transfer the supernatant to a fresh tube kept on ice. Discard the pellet.

2. Sample preparation

a.	Remove a small volume (50 μl) of lysate to perform a protein assay. Determine the protein concentration for each cell lysate.
b.	To the remaining volume of cell lysate, add an equal volume of 2x Laemmli sample buffer.
	*We recommend to reduce and denature the sample using the following method unless the product datasheet directs that non-reducing and non-denaturing conditions should be used.*
c.	To reduce and denature: Boil each cell lysate in sample buffer at 100°C for 5 minutes and aliquot. Store lysates at -20°C. Note: aliquot cell lysates (50-100 μl) to avoid repeat freeze/thaw cycles.
d.	Defrost tubes containing cell lysate at 37°C. Centrifuge at 16,000 x g in a microcentrifuge for 5 minutes.

3. Loading and running the gel

a.	Load equal amounts of protein into the wells of the SDS-PAGE gel, along with molecular weight markers. Load 20-30 μg of total protein from cell lysate or tissue homogenate, or 10-100 ng of purified protein.
b.	Run the gel for 1 to 2 hours at 100 V.
	*This time and voltage may require some optimization. A reducing gel should be used unless non reducing conditions are recommended on the product datasheet.*

4. Transferring the protein from the gel to the membrane

a.	Prepare the transfer stack and allow the protein to move onto the membrane. The membrane can be either nitrocellulose or PVDF; each has its advantages. “Activate” PVDF with methanol for one minute and rinse with transfer buffer before preparing the stack. The time and voltage may require some optimization. The membrane is ready for antibody staining.
	*Transfer to the membrane can be checked using Ponceau Red staining before the blocking step.*

5. Antibody staining

a.	Block the membrane for 1 hour at room temperature or overnight at 4°C using 5% blocking solution.
b.	Incubate membrane with appropriate dilutions of primary antibody in 5% or 2% blocking solution overnight at 4°C or for 2 hours at room temperature.
c.	Wash the membrane in three washes of TBST, 5 minutes each.
d.	Incubate the membrane with the recommended dilution of labeled secondary antibody in 5% blocking buffer in TBST at room temperature for 1 hour.
e.	Wash the membrane in three washes of TBST, 5 minutes each, then rinse in TBS.
f.	Let the signal develop, according to standard protocols.
g.	Remove excess reagent and cover the membrane in transparent plastic wrap.
h.	Acquire image using darkroom development techniques for chemiluminesence, or normal image scanning methods for colorimetric detection.

Western Blot - Troubleshooting

No signal

The primary antibody and the secondary antibody are not compatible
Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary).

Not enough primary or secondary antibody is bound to the protein of interest
Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC.

Cross-reaction between blocking agent and primary or secondary antibody
Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents are milk, BSA, serum or gelatin).

The primary antibody does not recognize the protein in the species being tested
Check the datasheet for species recognition.

Insufficient antigen
Load at least 20-30 μg protein per lane: Use protease inhibitors. Run the recommended positive control.

The protein of interest is not abundantly present in the tissue
Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.).

Poor transfer of protein to membrane
Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the wrong way; if using PVDF membrane make sure you pre-soak the membrane in methanol then in transfer buffer.

Excessive washing of the membrane
Do not over wash the membrane. Too much blocking does not allow you to visualize your protein of interest.

Over-use of the primary antibody
Use fresh antibody as the effective concentration is lowered upon each re-use.

Secondary antibody inhibited by sodium azide
Do not use sodium azide together with HRP-conjugated antibodies.

High background

Blocking of non-specific binding might be absent or insufficient
Increase the blocking incubation period and consider changing blocking agent.

The primary antibody concentration may be too high
Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best).

Incubation temperature may be too high
Incubate blot at 4°C.

The secondary antibody may be binding non-specifically or reacting with the blocking reagent
Run a secondary control without primary antibody.

Washing of unbound antibodies may be insufficient
Increase the number of washes.

Your choice of membrane may give high background
Nitrocellulose membrane is considered to give less background than PVDF.

The membrane has dried out
Care should be taken to prevent the membrane from drying out during incubation.

Multiple bands

The protein sample has multiple modified forms in vivo such as acetylation, methylation, myristylation, phosphorylation, glycosylation, etc.
Examine the literature and use an agent to dephosphorylate, deglycosylate, etc. the protein to bring it to the correct size.

The target in your protein sample has been digested (more likely if the bands are of lower molecular weight)
Make sure that you incorporate sufficient protease inhibitors in your sample buffer.

Unreported novel proteins or different splice variants that share similar epitopes and could possibly be from the same protein family are being detected
Check the literature for other reports and also perform a BLAST search.

Primary antibody concentration is too high - at high concentration multiple bands are often seen
Try decreasing the antibody concentration and/or the incubation period.

Secondary antibody concentration is too high - at high concentration secondaries will bind non-specifically
Try decreasing the concentration. Run a secondary antibody control (without the primary).

The antibody has not been purified
Try to use affinity purified antibody. This will often remove non-specific bands.

The protein target may form multimers
Try boiling in SDS-PAGE for 10 minutes rather than 5 minutes to disrupt multimers.

Uneven white “spots”on the blot

Air bubbles were trapped against the membrane during transfer or the antibody is not evenly spread on the membrane
Make sure you remove bubbles when preparing the gel for transfer. Incubate antibodies under agitation.

Black dots on the blot

The antibodies are binding to the blocking agent
Filter the blocking agent.

White bands on a black blot (negative of expected blot)

Too much primary and/or too much secondary antibody
Dilute the antibodies more.

MW marker lane is black

The antibody is reacting with the MW marker
Add a blank lane between the MW marker and the first sample lane.

The band of interest is very low/high on the blot

Separation is not efficient
Change the gel percentage: a higher percentage for small protein, lower percentage for large proteins.

Smile effect of the bands

Migration was too fast
Migration was too hot (changing the pH and altering the migration)
Slow down the migration or run the gel in the cold room or on ice.

Western Blot - References

Molecular Biomethods Handbook: J.M. Walker & R. Rapley (2^nd Edition); Humana Press, 1-1124 (2008)

Protein Blotting and Detection: Methods and Protocols: B.T. Kurien & R.H. Scofield; Methods Mol. Biol. 536, 1-588 (2009)

Western Blot - Online Resources

http://en.wikipedia.org/wiki/Western_blot

http://cshprotocols.cshlp.org/cgi/collection/immunoblotting

Western Blot: Technique, Theory, and Trouble Shooting: T. Mahmood & P.-C. Yang; N. Am. J. Med. Sci. 4, 429 (2012)

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