Western blot

From Molecular Biology Wiki

Western blot analysis can detect a specific protein in a mixture of any number of proteins while giving you information about the size of the protein. It is able to do this based on the use of a specific monoclonal antibody or polyclonal antibody. It does not matter whether the protein has been synthesized in vitro or extracted from cells in vivo or ex vivo. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. Thus, you must be able to produce at least a small portion of the protein from a cloned DNA fragment or purified protein which is then injected into a mouse or rabbit to generate a monoclonal antibody or a polyclonal antibody respectively. You will use this antibody as a probe to detect the protein of interest.

Western blotting tells you how much protein has accumulated in cells. If you are interested in the rate of synthesis of a protein, Radio-immunoprecipitation (RIP) may be the best assay for you. Also, if a protein is degraded quickly, Western blotting won't detect it well; you'll need to use (RIP). See the section on RIP for more information, as well as a helpful comparative chart that illustrates the differences between these two techniques.

Contents 1 Protein Sample Preparation for Western Blot 2 Western Blot and SDS-PAGE Electrophoresis 3 Membrane Transfer 4 Western Blot Buffers 5 Western Blot Protocol 6 Preparing samples for western blot 7 Loading Samples for SDS-PAGE | Western BLot 8 Running the SDS-PAGE Gel for Western Blot 9 References 10 Western Blot Forum Discussion

Protein Sample Preparation for Western Blot

Protease inhibitors

PMSF

Laemmli buffer ^[1]

Western Blot and SDS-PAGE Electrophoresis

SDS-PAGE

Membrane Transfer

Transfer buffer recipe

Western Blot Buffers

TBS buffer 10X TBS buffer stock solution

TBST buffer 10X TBST buffer stock solution

Western blot blocking buffer

Western Blot Protocol

A detailed western blot protocol.

Preparing samples for western blot

• Obtain sample loading buffer (Laemmli buffer) ^[2].
• Add 20 ul of 1X sample buffer to 20 ul of sample (or load equal concentration of protein).
• Boil samples on heater for 5 - 6 minutes at 90-100 C.
• Centrifuge samples after boiling. (this spins down any condensation)

Loading Samples for SDS-PAGE | Western BLot

• Pour running buffer into the middle of the running apparatus.
• Fill ONLY running buffer upto the bottom of the glass plates. (this prevents current leakage from the apparatus from cracks and defects)
• Load Protein marker (also called protein ladder) (in different lanes when running several gels to differentiate different gels from each other)

Running the SDS-PAGE Gel for Western Blot

• Run gel at 100V to 110 V for quick running OR for better resolution run gel at 35 - 40 mA (Constant current) in order to get sharp bands. This takes 2 - 2 1/2 hours to run.

• Bubbles should be seen if the apparatus is working well.
• Check in 5-10 minutes for migration of protein ladder downwards.

References

1. ↑ Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, (1970)
2. ↑ Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, (1970)

See Also:

• Western Blot Background
• Western Blot

Western Blot Forum Discussion

Join the specifc discussions on Western Blot! Or simply click on the top (Discussion) link to talk in general about Western Blot!

		Thread / Thread Starter	Last Post	Replies	Views	Forum
		Anyone treating Cell lysates with RNAse/DNAse danfive	05-15-2008 01:22 PM by admin	1	71	Western Blot Forum
		Fast Westerns gosens08	05-14-2008 02:39 PM by radioheadhead	6	349	Western Blot Forum
		negative band on film alexr	05-13-2008 08:33 AM by kmunson779	1	35	Western Blot Forum
		PBS in fractions Jasmina	05-13-2008 05:30 AM by alexr	3	133	Western Blot Forum
		Sample Buffer abelaksono	05-02-2008 05:07 AM by abelaksono	2	101	Western Blot Forum
		p-ERK.. Bouchra	04-30-2008 11:18 AM by admin	3	81	Western Blot Forum
		Western Blot Background Troubleshooting molecule2005	04-15-2008 01:21 PM by pabells	9	1,043	Western Blot Forum
		internal control for plant proteins in WB izacil	04-09-2008 09:21 AM by admin	1	157	Western Blot Forum
		Loading control? Labnovice	04-07-2008 10:22 AM by kmunson779	1	217	Western Blot Forum
		HRP marker genetherapyTNPRC	04-01-2008 03:34 PM by danfive	1	123	Western Blot Forum