Western Blotting

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Western blotting, or immunoblotting, is a widely used and powerful analytical tool used to detect specific proteins, or protein modifications, in a sample of tissue/cell homogenate or extract. There are numerous variations of western blotting equipment and reagents that allow for detection and measurement of a multitude of proteins with a wide array of molecular properties.

 

What is Western Blotting?

 

In 1979, Towbin, et al., introduced a new, previously unknown technique to separate and identify proteins, and called it western blotting1. This technique allows for the separation, detection and measurement (qualitative and quantitative) of specific targets, through antibody/antigen interactions, within a complex biological sample such as cell and/or tissue lysates. Western blotting, or immunoblotting, can be used to detect proteins and post-translational modifications (ex. phosphorylation) as well as confirm gene expression, and is now a mainstay of most research labs.

 

How Does It Work?

 

Western Blotting - How Does It Work?The first step in western blotting is to separate the proteins contained within the sample, which is done using gel electrophoresis. Biological samples are loaded into a gel and electricity is passed through allowing the separation of proteins by molecular weight. Molecular weight is inversely proportional to migration in a gel; therefore the smallest proteins travel the furthest down the gel. Protein “ladders” that contain dyed proteins can be loaded at either end of the gel to illustrate where specific molecular weights will travel down the gel to aid in the identification of the specific target of interest. The proteins contained within the gel are then transferred, or blotted, out of the gel and onto the surface of a membrane (ex. PVDF or nitrocellulose). To prevent non-specific antibody binding, the membrane is incubated in a protein blocking buffer, and then probed with a primary antibody specific to the protein of interest. After which, the membrane is incubated in a secondary antibody that is specific to the host species of the primary antibody. The secondary antibody is conjugated to a reporter molecule that allows the detection of the antibody signal. Between each incubation period the membrane is washed with a buffer containing a detergent to wash any non-bound antibodies from the surface of the membrane.

There are several varieties of reporter molecules and hence detection methods available. Colorimetric detection can be produced by using horseradish peroxidase or alkaline phosphatase conjugated to the secondary antibody. Colorimetric substrates produce a precipitate that is deposited on the membrane and is visible to the naked eye. Currently the most sensitive and popular detection method uses chemiluminescent substrates. The reaction with the enzyme conjugated to the secondary antibody produces light as a byproduct and can be captured with film. The light output can also be measured with digital imaging based on charge-coupled device (CCD) cameras, which are gaining in popularity as an alternative to film for capturing the signal. In addition, fluorescent tags can also be bound to secondary antibodies but require an instrument capable of capturing fluorescent signals. Western blotting using fluorescence is a somewhat newer technique that allows for some interesting potentials, including the possibility of multiplexing that would allow for the measurement of multiple targets on a single blot without stripping and re-incubating in antibodies. Whichever method of detection is used, the amount of protein on the blot should correlate with the intensity of the signal obtained.

 

Why Western Blotting?

 Western Blotting - Why Should We Use It? 

Western blotting is a vigorous research tool that allows scientists to detect and, with specific methods, measure the amount of protein present in a sample. Experimentally, western blotting can be used to measure and compare expression of proteins, post-translational modifications and gene expression in control and experimental tissues or cell lysates. In the clinical setting, western blotting can also be used to examine differences in protein expressions between healthy and diseased tissues.

 

Recent Western Blotting Citations Using Bethyl Antibodies

 

Bethyl Laboratories sells high quality Western blot antibodies. These products have recently been used to study:

 

  • How human cells respond to DNA damage caused by UV radiation2
  • The relationship between the stress-response and global protein translation in stem cells3
  • Improving recovery after brain-related disease, such as stroke or brain tumors4
  • Hundreds of RNA-binding proteins associated with cancer, neurobiology, and development5
  • Regulation of cellular communication and renewal in the intestinal tract6

 

 

Thousands of Bethyl antibodies are validated for use in western blotting. The complete list is here:

 

WB

A20
AC9
ACD
ACL
ACS
ADA
ADK
AE2
AF4
AF6
AF9
AIF
AIP
ALB
Aly
AP4
AQR
AR
ARG
ASF
ATM
ATR
AU1
AU5
Axl
AZ1
C3
C3G
CAD
CAL
CAR
CAT
CBL
CBP
CBS
CD4
CD5
CD7
CEE
CGN
CHC
CIA
CIC
CIT
CIZ
CP
CPD
CRP
CSB
CSK
Maf
MAX
MAZ
MB
MFF
MK5
MNT
MOF
MVP
MYB
MYC
p17
P23
p27
p49
p53
P55
p66
p73
p75
PA1
PAF
PAM
PB1
PBE
PBK
Pc2
PC4
PDI
PF1
PGD
PGP
PGR
PHB
PKM
PML
PNN
PNP
PPL
PSF
PXN
PZR
RAN
RCL
RCP
REA
RED
REL
RIG
RLF
RMP
ROD
RP1
SA1
SA2
SCD
SET
SF1
SFN
SIL
SIP
SKI
SKT
SLK
Sp1
SPN
SPP
SRA
SRF
SRM
SSB
SYK
T7
TBP
TDG
TF
TFG
TfR
TG2
TK1
TPR
TR2
Trf
TS
TSN
TSP
TTK
TZF
References:

1. Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PNAS. Sept 76:4350-4354.

2. Harley ME, Murina O, Leitch A, Higgs MR, Bicknell LS, Yigit G, Blackford AN, Zlatanou A, Mackenzie KJ, Reddy K, et al. 2016. TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism. Nat Genet. Jan:48(1): 36-43. [Bethyl antibodies used: Phospho SMC1 (S966) Antibody (A300-050A), Phospho RPA32 (S4/S8) Antibody (A300-245A), Phospho KAP-1 (S824) Antibody (A300-767A)]

3. Blanco S, Bandiera R, Popis M, Hussain S, Lombard P, Aleksic J, Sajini A, Tanna H, Cortés-Garrido R, Gkatza N, et al. 2016. Stem cell function and stress response are controlled by protein synthesis. Nature. Jun 16;534(7607):335-340. [Bethyl antibodies used: Claspin Antibody (A300-266A)]

4. Karuppagounder SS, Alim I, Khim SJ, Bourassa MW, Sleiman SF, John R, Thinnes CC, Yeh TL, Demetriades M, Neitemeier S, et al. 2016. Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models. Sci Transl Med. Mar 2:8(328):328-329. [Bethyl antibody used: SMC3 Antibody (A300-060A)]

5. Sundararaman B, Zhan L, Blue SM, Stanton R, Elkins K, Olson S, Wei X, Van Nostrand EL, Pratt GA, Huelga SC, et al. 2016. Resources for the comprehensive discovery of functional RNA elements. Mol Cell. Mar 17; 61(6): 903-913. [330 Bethyl antibodies were used; please refer to the article for the complete list]

6. Imajo M, Ebisya M, Nishida, E. 2015. Dual role of YAP and TAZ in renewal of the intestinal epithelium. Nat Cell Biol. Jan:17(1):7-19. [Bethyl antibody used: LATS1/LATS2 Antibody (A300-479A)]