Technique / Proteomics and protein biochemistry / Western blot assay


Western blot protocol



Western blot protocol

Preparation of cell lysates from E.Coli bacteria for western blotting:

1. Spin down 1 ML overnight culture
2. add 100-200 ul BugBuster protein extraction reagent (Novagen cat. No. 70584)
3. Sonicate 1 min
4. add 2x sample buffer to same lysis volume(or add sample buffer directly to the cell pallets)
5. Boiling for 5 min

Preparation of PVDF membrane for the blotting step:

1. Cut a piece of PVDF membrane(Bio-Rad Immun-Blot PVDF Membrane Cat. No. 162-0177)
2. Wet membrane for 30''-60" in Methanol
3. Transfer membrane to 1x Transfer buffer( Life gels LongLife Transfer Buffer 20x Cat.No. BG-168) untill use.

Use precast gels (Ready gel from Lifegels Cat. No.NH21-420 or BioRad cat. No.161-1104) for western blot protein electrophoresis:

1. Assemble gel in gel tank(BioRad Mini-PROTEAN 3 Cell cat. No. 165-3301 165-3302)
2. Load 10-30 ug protein each lane.
3. Use 8-15 ul protein marker.
4. Run at 100V-150V(constant voltage) for 50 min-1.5 hour (use Hepes-Tris running Buffer)

Western blotting membrane Transfer:

1. Prewet the sponge, filter papers(Biorad cat. no.1703932) in 1X Transfer buffer
2. Assemble "sanwich" in BioRad transblot cat no.170-3930 170-3935):
Black (negative charge)-sponges, filter paper-gel-membrane-filter paper-sponge-red (positive charge)
3. Transfer at 200-250 mM(around 100V) for 1-2 hour with cold pack or pre-chilled buffer. Larger protein take longer time to transfer.

4. When blot transferation completed, take membrane out if transfer buffer contain 20% methanol, (or soak in methanol again about 15") let it completely dry in RT.(over 30 min)
5. Wet membrane in methanol 15 seconds, and resin in ddH2O.
6. Add in 5 % Bloking buffer (5 g dry milk, BioRad cat.no.170-6404 in TBS 0.1% tween-20) and block overnight 4C or 1-2 hour RT.

Western blot antibody detection:

1. Incubate with primary antibody diluted in 1% Blocking buffer in TBS-T 0.1% tween-20) for 1-2 hour RT, or overnight @ 4c.
2. Wash blot 3x 10 min with 0.1% Tween 20 in TBS.
3. Incubate blot with Secondary antobody (HRP conjugated anti rabbit or mouse IgG) 1:10000 in 1% Blocking buffer 0.1% Tween-20 TBS for 45min-60min.

4. Wash blot 3 X 10 min with 0.1% Tween-20 in TBS.
5. Detect with Amersham ECL Plus kit (RPN 2132) or other ECL detection kits. Bring ECL plus kit to room temperature. Make solution A:B 40:1 mix. Add 2ml to 1 membrane for 5 min. wrap with film for either film develop or camera picture. Do no let membrane dry.

Western blot stripping protocol:

1. Rinse blot with 0.1% Tween 20 in TBS.
2. Add Strpping buffer 37 C 30 min(Pierce cat.no.21059)
3. Rinse blot with 0.1% Tween 20 in TBS.
4. Start again with membrane blocking step and re-probe with new antibody.

Selected online western blot protocols:

Western blot protocol (University of Washington, Department of Pathology)
Western blotting protocol with reagent and buffer (Frank lab, Oklahoma Medical Research Foundation)
Western blot protocol with buffer recipes (Howell lab, UCSD cancer center)

Last update 27-Aug-2006, Rating of 11 votes.

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By Javid Mohammed on 16-Mar-2009
This is reply for FEVERA's question:

Hi! You could use Precision Plus Protein Western C Standard from Biorad (Cat# 161-0376). The advantage of using this standard is.. you could not only see the protein migration while electrophoresis, but also could visualize it on the blot by ECL detection (NOTE: for ECL detection, you also need Streptactin-HRP conjugate from Biorad, that you use during secondary probing). Also, yes, you need to be careful that you do not use Streptactin-HRP in excess... the chemiluminiscence of your standard could outstand your desired signal (esp the sample in the lane next to the ladder), b'cuz of which you can't expose your blot to the film for longer time when required (when the signal from your desired protein is low...). Biorad recommends using 1ul of Streptactin-HRP in 10ml. I generally use 0.1ul in 10ml which still sometimes imparts excess signal. So, I would recommend, in case if you are gonna use this.... Use 4ul of Standard, and 0.1ul of Streptactin-HRP/10ml.....

Have any further queries...Feel free to shoot me an e-mail,...

Best regards,
Javid
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By Javid Mohammed on 16-Mar-2009
This is a reply for Dr. Satwinder Kaur:

Hi Dr Kaur. There are basically a couple of approaches for the visualization of desired proteins on western blot; Chemiluminiscence and Flourescence....

Flourescence Approch: Should use flourochrome labeled (Infrared Dye) primary (direct method) or secondary (indirect method) antibodies. Infrastructure needed: Florescence detection system such as LiCOR. Advantage of this approach: You could visualize multiple proteins on the same blot with different IR-Dye labeled antibodies...(thus, you do not have to strip a blot and reprobe).. You could save on Photographic film and Chemiluminiscent detection reagents...

Chemiluminiscense approach: Here you use antibodies such as HRP-labeled that generate chemiluminiscence upon reaction with its specific substrate.... The most popular of one such kits is ECL Western detection kit from GE (Amersham). Here you also need either a Chemiluminiscence gel documentation system or Photographic film + Developing equipment.... Advantages: HRP-Antibodies are less expensive that IR-Dye labeled antibodies.. Disadvantages: Strip the blot and reprobe for each desired protein to be analyzed... Expenses with ECL detection reagent and film....
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By agass on 16-Dec-2008
Two methods of electroblotting proteins to membranes, wet and semidry protein transfer. Semidry blotting is quicker and uses much less buffer but wet blotting is more effective if the proteins of interest are of high molecular
weight (>100 kDa)
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By Dr.Satwinder Kaur on 17-Sep-2008
Kindly let me know the basic infrastructure required for the visualization of the western blots?or what are the techniques avaible for the detection of the blots?
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By Ceci on 21-May-2008
hi! I need to know if is better not to use SDS in the transfer buffer while using a PVDF membrane.

Thanks!
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By shahoo on 14-Apr-2008
i want to know is it possible to use towbin transfer buffer(tris,glycin,methanol) instead of ssc buffer to transfer my protein on nylon membrane positively charged(roche) to do western blot?
thank you for your help
sincerely yours,shahoo
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By reena on 13-Jul-2007
i am able to see a band of exactly 14.5 Kd in my SDS PAGE. it appears some times and dose not appear some time with silverstaining. it never appears with coomasie. if i do a westernblotting of that i am able to detect it with the same antibodies as that against my protein which is 18.6Kd protein. i am not able to judge whether it is an artifact or is it a real band chopped off from my protein? what could be the possible reason of it being chopped off in such a way if true.and if so the case why am i not seeing it every time i run the same sample.
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By Cytoplasm on 10-Jul-2007
Alkaline peroxidase labels work well with chemoluminscent reagents in western blot. Substrate for western blot detection is the key for high sensitivity and various vendors offer different kits for this purpose.
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By fevera on 26-Mar-2007
hi, our lab is using HRP-conjugated antibodies for western blot detection and is using multi-color protein ladder (protein marker). We want to switch to HRP-labeled protein marker, can anyone recommend a supplier? Normally how much such starndard ladder is needed in a mini gel? And finally, does it cause any extra background compared with multi-colored marker? Thanks!
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By western blot on 04-Mar-2007
Four basic procedures of western blot:

1) Gel electrophoresis (SDS-PAGE, non-denaturing PAGE etc) protein separation.

2) Transferation of proteins from gel to membrane (blotting step) such as nitrocellulose, nylone, PVDF etc.

3) The blotted membrane is then incubated with blocking buffer containing generic protein to block the non-specific binding sites on the membrane. Membrane is then incubated with primary and secondary antibody for protein of your interests.

4) Detection and documentation of signals revealed by enzymatic reaction associated with the secondary antibody after adding specific substrate.

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By Alan on 27-Aug-2006
Is it necessary to dry membrane completely and re-hydrate it with methanol again after each membrane transfer? What is the benifit of doing this? Thanks. --alan
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