Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 
Visit old site
Home Print this page Email this page Small font size Default font size Increase font size
Users Online: 636


 
 Table of Contents  
LETTER TO EDITOR
Year : 2014  |  Volume : 6  |  Issue : 3  |  Page : 158-159

Western blot: Protein transfer overview


Department of Life Sciences, Anglia Ruskin University, Cambridge, United Kingdom

Date of Web Publication11-Mar-2014

Correspondence Address:
Janay Gibbons
Department of Life Sciences, Anglia Ruskin University, Cambridge
United Kingdom
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1947-2714.128481

Rights and Permissions

How to cite this article:
Gibbons J. Western blot: Protein transfer overview. North Am J Med Sci 2014;6:158-9

How to cite this URL:
Gibbons J. Western blot: Protein transfer overview. North Am J Med Sci [serial online] 2014 [cited 2019 Nov 19];6:158-9. Available from: http://www.najms.org/text.asp?2014/6/3/158/128481

Dear Editor,

The article entitled 'Western Blot: Technique, Theory, and Trouble Shooting' was helpful and provided a detailed protocol for all stages of Western blotting. [1] However, there are concerns regarding [Figure 1], which depicts the submersion of an electrophorator including power leads, in an ice water bath. This arrangement is a serious health and safety hazard due to the posed risk of electric shock and is therefore not recommended.

Although Western blotting involves passing an electric current through a liquid environment, it is not a dangerous technique if performed properly; however, it is easy to overlook the risks of each stage. Protein extraction often uses β-mercaptoethanol and sodium dodecyl sulfate (SDS), both of which are corrosive, acutely toxic and the former mutagenic. To limit risk they should be handled with care, in a fume hood with the use of personal protective equipment. Polyacrylamide gel electrophoresis (PAGE) is only unsafe if the equipment is not used correctly. Some tanks allow the unit to be plugged into the power pack without covering the buffer. The buffer should be covered at all times when attached to the power supply to prevent contact with the electrified liquid. The protein transfer stage should carry equal risks to PAGE, although many labs adopt the method depicted in [Figure 1] of the aforementioned article, [1] which introduces further possibility of electric shock.
Figure 1: A submersed electrophorator. Figure 7 from 'Western Blot: Technique, Theory, and Trouble Shooting'.[1]

Click here to view


There are many safer methods for transferring proteins to a membrane. [2] Electroelution (wet, semi-dry, and dry) and diffusion transfer techniques all have their merits and applications, but it is important to note that method alone will not guarantee a successful blot. Effective protein transfer is also heavily reliant on the gel acrylamide percentage, the molecular weight of electrophoresed proteins, and the blotting membrane used. [3] A number of membranes have been developed in the past, but only nitrocellulose and polyvinylidene fluoride (PVDF) remain popular. PVDF offers a higher binding capacity than nitrocellulose, [4] but likewise has higher background binding. [1] PVDF is also physically stronger than nitrocellulose, [4] so may be preferable for stripping and reprobing.

The diffusion method is not widely utilized as it provides qualitative rather than quantitative transfer, [2] but is especially useful in producing up to 12 blots per SDS-PAGE gel for screening with multiple antibodies. [5] The technique involves sandwiching a single gel between two membranes and clamping it between glass plates to facilitate diffusion. [2] The process is repeated up to six times to produce up to 12 blots, as desired. [3]

Dry transfer is becoming increasingly popular due to the Life Technologies' iBlot® systems, which boast a blotting time of only 7 min. The company claims their iBlot® systems produce superior protein transfer quality in comparison to wet and semi-dry methods. [6] Little data currently exists to support these claims.

Semi-dry transfer involves soaking up to six layers of gel, membrane, and filter paper in transfer buffer and sandwiching them between two horizontal plate electrodes. [7] It is slower than dry blotting, but is generally more rapid and efficient than wet transfer, [8] and is especially suited to low molecular weight proteins. [3] Other advantages over wet transfer include more cost efficient electrodes, less complicated power packs, and the ability to blot several gels simultaneously. [4] [Figure 2] shows the final product of western blotting using semi-dry protein transfer.

Wet transfer involves immersing a gel-membrane sandwich in an upright tank of transfer buffer, usually with vertical platinum electrodes. [2] The 'conventional' wet transfer technique employs the use of a cooling system to reduce heat produced by the electrodes, although over the years modifications have been developed. One modification, the heat-mediated blotting method, is preferable over the conventional protocol due to enhanced transfer of both high and low molecular weight proteins. [9] The heated buffer is thought to increase permeability of the polyacrylamide gel, thus facilitating faster protein transfer. [3] Still if use of the 'conventional' wet transfer method is desirable, there are a number of companies that offer specialized wet transfer equipment. These include built-in cooling systems that eliminate the need for ice immersion. If this equipment is unavailable, the tank can be wrapped in ice packs and placed into a cold room, otherwise if an ice bath is absolutely necessary, ensure the level of ice is below that of the electrical components.
Figure 2: A western blot of collagen-induced platelet activation over time using semi-dry protein transfer

Click here to view


Successful protein blots can be produced via a range of methods, all of which have advantages and disadvantages, yet regardless of the technique, safety should always be a priority.

 
  References Top

1.Mahmood T, Yang PC. Western Blot: Technique, theory, and trouble shooting. N Am J Med Sci 2012;4:429-34.  Back to cited text no. 1
    
2.Kurien BT, Scofield RH. Western blotting. Methods 2006;38:283-93.  Back to cited text no. 2
    
3.MacPhee DJ. Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods 2010;61:171-7.  Back to cited text no. 3
    
4.Kurien BT, Scofield RH. Protein blotting: A review. J Immunol Methods 2003;274:1-15.  Back to cited text no. 4
    
5.Kurien BT, Scofield RH. Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens. J Immunol Methods 1997;205:91-4.  Back to cited text no. 5
    
6.The iBlot® Dry Blotting System vs. Conventional Semi-Dry and Wet Transfer Systems. Paisley, UK. Life Technologies, 2013. (Accessed December 17, 2013, at http://www.lifetechnologies.com/uk/en/home/life-science/protein-expression-and-analysis/western-blotting/western-blot-transfer/iblot-dry-blottingsystem/iblot-dry-blotting-comparison-to-semi-dry-and-wet.html).  Back to cited text no. 6
    
7.Kurien B, Scofield R. Introduction to Protein Blotting. In: Kurien B, Scofield R (Eds.), Protein blotting and detection. New York: Humana Press 2009;3:9-21.  Back to cited text no. 7
    
8.Wisdom GB. Protein blotting. Methods Mol Biol 1994;32:207-13.  Back to cited text no. 8
    
9.Kurien BT, Scofield RH. Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes. J Immunol Methods 2002;266:127-33.  Back to cited text no. 9
    


    Figures

  [Figure 1], [Figure 2]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
References
Article Figures

 Article Access Statistics
    Viewed2124    
    Printed32    
    Emailed0    
    PDF Downloaded649    
    Comments [Add]    

Recommend this journal