Detection of large protein molecules

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xiancai zhong

Thursday, 28 Jun 2012 06:31 UTC

Hello

I have a trouble to detect the protein MUC2 by normal western blotting via SDS-PAGE electrophoresis. 
I saw someone use SDS-agrose gel or gradient PAGE gel to do electrophoresis. But there isn’t anybody who has such experience in our lab.
Is there anyone who detected this protein or other big molecules?
Please share you expience to us. 
I really want to know how to detect such molecules with high molecular weght. 
Thaks a lot.

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Shivasankari Gomathinayagam

04 Aug 2012 | 19:21

It is usually difficult to transfer high molecular weight proteins ! You can try the following :

1. Increase the amount of methanol in the transfer buffer.
2. Transfer o/n in +4.
3. Make sure your lysis buffer is right, we had a similar issue in the lab and then playing around with the lysis buffer did help !
4. Are you sure about your Ab ? In most of our cases the problem is from the Ab !

Hope this helps !

ALL the Best !

SS

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xiancai zhong

10 Aug 2012 | 04:33

Hello
Thank you for your kindness, but, can you explain it in detail?
1. The percentage of methanol in our transfer buffer is 20%. If I increase the amount of methanol, how much should use? 
2. What does +4 mean? 4 degree?
3. I used the lysis buffer from cell signaling companay. I dilluted 10X lysis buffer using distilled water adding PMSF and EDTA free protease inhibitor. Is it OK?
4. I used two different Abs. I am not sure it is the problem of Abs, because I couldn’t get any band.
5. What did you use to detect large protein? SDS-PAGE or SDS-agarose gel?

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Luka Mihajlovic

19 Aug 2012 | 18:24

Hi, 
How big is your protein? I worked with composite gels with very large (several MDa… )protein crosslinks, and, except for the fact that they are very manually demanding (as in flimsy as hell), I had no problems with the transfer step – everything worked pretty much normally. Check your antibodies.

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xiancai zhong

25 Aug 2012 | 02:07

My protein is 600kDa. And usually it forms oligomers that is much bigger. But the 2-mercaptoethanol is always applied in western blot. I think it is possible to get the band of monomer, right?

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Safoura Sameni

24 Aug 2012 | 04:19

Hi Zhong,

You might want to check for couple of more changes:

1-Use a 5% gel.
2-Change your transfer condition to 90V for 3hrs.
3-Change the ice pack every one hour, and it’s better that the transfer been done with cold transfer buffer in cold room or in an ice box.
4-Make sure that your APS is new.

Good luck

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Kellis Rolland

16 Jun 2013 | 12:38

Thank’s, very informative!