Immunoblotting - (Nov/13/2009 )

Hi!!
I have been trying to do an immunblot analysis of collagen type I purified from different organisms. I can see the bands clearly on SDS-PAGE. But my developed blot looks as if I have done some negative staining. The band of my interest is not satined but the background has got stained. Can any body tell me what could be the problem.. What would the ideal protein concentration be??
Please enlighten me!!! I am getting frustrated with this excercise!
Minni

hey minni we can help better if u can give in more details like wat membrane u use... wat voltage u run at for wat time??!! wat is ur loading amount.. is it a reduced sample.. wat kind of detection system u are using etc..

Minni on Nov 13 2009, 01:14 AM said:

Usually it's just because there's overload of protein which appears negatively on the stained background ! Do you use HRP or AP labelled antibodies ?

Pradeep Iyer on Nov 13 2009, 03:17 PM said:

Hi!! Pradeep, It will be really nice!!
Following are the details:
1. The protein I am trying to detect is collagen.
2. The membrane I use is nitrocellulose.
3. I run the Gel at 80V till the dye front enters separating then at 100V.
4. Gel percentage is 7%.
5. Transfer is done at 50mA over nnight in a wet transfer apparatus in towbin's buffer containing Tris glycine and methanol.
6. The detection system I use is AP conjugated secondary antibody. NBT-BCIP system.

I have a lot constraints. I am working for a very small company in CHennai, India. There is practically no budget for R&D. So, I connot go in for ECL based detection. We cannot afford to buy our reagents from bigger companies.. Therefore the reagents I am using are from local Indian companies.... Buffers etc I prepare my self.. The primary antibody is from Santacruz.. Secondary is from Bangalore genei.
Is there anything else you would like to know??
I am really stuck actually.
Thankyou
Meenakshi/minni

Hi meenakshi..
perfectly fine. i m in india and i know the constraints!!! (which lab do you work for by the way??)
your antibodies and detection systems are great... the flaw might be running a wet transfer for overnight.. tat might just be too long a time for a blowthru of your protein.. also the gel percentage is less (7%) so i think such a long blot time is not required...
we run the sds gels at 130 V constant till the dye elusion.. we use PVDF.. but NC shud be no problem (dont prewet it with methonol) although do prewet it with the blot buffer
do a western blot with 100V constant for one hour keeping 400 mA ax current. this shud work out.. generally bangalore genieee antibodies (Secondary) work well in a 1:2000 dilution!!
Good luck!!

Hi!! Pradeep
Thankyou very Much for suggestions. I will definitly try them out on monday.
I work for a company called Eucare pharmaceuticals Pvt. Ltd. I am supposed to be the head of R&D here. I have done my ph. D. From JNU Delhi and did post doctoral work in CLRI, CHennai. I must have run count less westerns.. But most proteins I have studied were in the range of 30-80 KDa. Collagen immunoblotting is first time experience for me.. and it is giving trouble as no other blot or protein has given me...!!!
Where are you from??
Meenakshi

Pradeep Iyer on Nov 14 2009, 11:12 AM said:

oh then i shud be calling u madam!!
i have done my post grad in biotech from madras univ... grad from xaviers.. mba from nim... worked in intas biopharma in ahmedabad, gujarat for 3 years.. now i m working for a new lab set up in hetero biotech, hyderabad.. i work in the analytics!!
good to know u!!!
keep in touch.. thanx for teh details.. not all give in so easily...

collagen is high molecular weight. you may want to try adding up to 0.05% sds and 20% methanol in the transfer buffer to facilitate transfer.

have you tried (can you try?) primary antibodies from a different source? some complain that not all santa cruz antibodies work as expected.