Researchers,
can anyone suggest me western blotting technique?
I need to perform western blotting but i have no practical experienced yet.
How to develop the band or expression of protein on membrane?
Will it appear expression of protein after primary and secondary antibody incubation or anythings else?
Please kindly suggest simple and accurate procedure for Western Blot.
Hi, I recommend you to read protocol first. As western blot has multiple steps, from protein isolation, gel preparation, loading, running, transfer, blocking, antibody incubation and finally detection.
So, it would be better if you can understand whole process first, then you can troubleshoot for your target protein detection according to your desire.
Here is something to read (no affiliation :)
http://www.bio-rad.com/LifeScience/pdf/Bulletin_2895.pdf
http://www.bio-rad.com/LifeScience/pdf/Bulletin_2895.pdf
Hi
find attached a protocol I regularly use. Some of your particulars might vary but at least this will give you an example of a WB SOP which in conjunction with Ducos background reading material will give you a start:
Western blotting rigs for electrophoresis and subsequent transfer of protein to NC or Protran membrane can vary in terms of voltage/current for both steps. Thus I have provided specific protocols for 2 of the most common sets of apparatus namely from Stratagene (x module) and Bio rad. It also assumes chemo luminescent detection with Amershams ECL. There are digital alternatives or indeed digital syterms can now be used to scan chemo luminiscent detection film such as Amershams hyperfilm
In addition, the protocols I have provided assume use of precast (Novex) polyacrylamide gels for intial electrophoresis
You can of course make your own gels but that adds to the complication, especially for somebody who is a complete beginner
You also need to consider production of protein lysate from tissue or cells to load onto your polyacrylamide gels. To that end I have attached an outlook message with basic protocols, background notes and some general guidance embedded
I will just now add one or 2 general but key points based on experience:
1. Load anything from 1ug to 10ug of total protein lysate per lane when detecting proteins using Amershams (GE Health care) ECL plus or 5ug to 30ug if detecting by the original and standard ECL formulation
2. For high copy number proteins go towards the lower amounts and for low copy number proteins the higher amount
3. I would recommend having performed PAGE to separate protein bands transferring to Protran/NC for 90 min -3 hours with your transfer rig either in a cold room with Methanol transfer buffer stirring or on ice. For proteins < 50kD in general 1 hour will give you efficient transfer whereas for proteins > 50kD and especially for larger proteins approximating to 100kD transfer for nearer to 3 hours
4. Having transferred make sure all of your protein marker is gone from your (dehydrated) acrylamide gel and can be seen on your membrane. This is a good way of telling whether your actual protein from your lysates have transferred successfully
5. Block your membrane in Tris buffered saline + 0.1% Tween 20 (TBST) + 3% BSA for 1 hour at room temp
6. Then when incubating in primary (epitope specific) ab dilute in TBST + 3% BSA (the above) and incubate over night at 4C for most proteins and for 1-2 hours at room temp for highly expressed proteins, e.g. structural or housekeeping proteins: Best signal to noise is obtained by diluting antibody in TBST at the lowest recommended concentration over night at 4C rather than a higher concentration for 1-2 hours at room temp. Thus by way of example, most primary antibodies are diluted between 1/50 and 1/1000 and for a typical antibody this could be 1/100 to 1/500. Therefore initially try 1/500 over night @ 4C
7. Please note: If you do elect to go down the chemo luminescent route and use Amersham's ultra sensitive ECL plus or ECL prime you can get away with using 1/5 to 1/10 x less compared to the recommended dilution on the data sheet. Thus, if the sheet says 1/100 for standard ECL in my experience you can use 1/500 or 1/1000 with ECL prime or plus
8. Having incubated with primary antibody over night wash 3 - 6 x 5 min with TBST (no BSA) and then incubate with the lowest recommended amount of conjugated secondary antibody for 1-2 hours @ RT. This will typically be a much lower dilution than your primary antibody, viz 1/1000 to 1/5000 and thereagain if you are using ultra sensitive chemoluminescent detection systems like ECL plus or ECL prime you can reduce that figure to 1/5 to 1/10 x that amounts in my experience
9. Wash as before then detect
During the whole process it is important to keep your membrane moist
Also when incubating with antibody or during washes keep everything moving either by rocking your membrane in a container or placing within a 50ml Falcon tube and place on a rolling device (my preferred choice)
If you encounter high background you can increase the tween 20 in your wash solutions to 0.5% (although keep at 0.1% when diluting your antibodies)
Other than that the main thing affecting Westerns is the source of antibody so in the past it hasn't been uncommon for me to try 1 type of ab - obtain multiple bands - and then repeat the blot using an alternative antibody and find I obtain my 1 specific band. Hurray !!!
Finally as you are a complete beginner I have now decided to attach a list of products you typically need for WB: I am not specifically recommending those companies (and let me say I have no affiliation to any company) but it gives you an example of what types of reagents you could use
Good luck !!!
Very good story of Laurence.
May I add to point 4 that you can check your loading at this point w/ e.g. ponceau red for NC or coomassi BB for protran.
Point 5/6. I would rather block o/n and keep the Ab-incubations quit short. That gives me less back grount signal.
- Badges
- Science method