Hi! Im performing a westren blot, but my immunoblotting isnt going well. The proteins transfer perfectly on the PVDF membrane, but when I incubate the membrane with my primary abs, the signal generated is like a torch light. The whole membrane lights up like a christmas tree!
I use a primary ab dilution of 1:500 which ive diluted to 1:1000 to check, I also use a secondary dilution of 1:500 which ive diluted further as well. Incubations with the primary is over night. I use santa cruz abs. The problem tho is when i apply the luminol substrate everything lights up, please help!
Hi,
perhaps your primary Ab recognizes a protein present in milk? ;)
Check Ab info card for compatibility with milk.
Check milk composition (e.g. http://www.emeraldinsight.com/journals.htm?articleid=866460).
Try blocking in 2-3% BSA in TBS-Nonidet or PBS-Nonidet.
Of course perform proper membrane washes, as suggested by Bharat!
Best,
Lukasz
try to decrease the amount of proteins u load...at least 2-3 times...u can try by loading your sample in different amount on the same gel/blot...we u will add the luminol u should find the optimal amount of sample to load!!!I would try also to dilute the secondary ab...as well as dilute the luminol solution with pbs, or water..WB is not a procedure..is an art!!!!;-)
good luck!
Thanks marco :) ive diluted the secondary already, will let you know in about 12 hours how that goes :)
The whole membrane lights up! The blocking solution i Use is 5% skimmed milk. I block for 1.5 hours after transfer. The transfer is fine, the markers appear beautifully on the PVDF and the gel is clear. I transfer in lots of ice to make sure ;hot' transfers dont happen. However, immunoblotting is an issue. I use 1:1000 1ary and 2ary abs. incubation with 1ary is overnight at 4 degrees and 2ary is 2 hours. I mix 1ml each of reagent A and B supplied by santa cruz (Luminol reagent) and apply it on the membrane, it then lights up like a LCD screen. So what should I be doing? I plan on diluting the 2ary to maybe 1:10,000 but other than that I dont know where to start.
I hope you rigorously wash your membrane with PBST/ TBST atleast 5 times 5' after primary Ab at RT rocking and then 5 times 10' rocking each after 2ndary. Is this overexpressed protein in bacteria or overexp protein in some cell line? If so load less or transfect less respectively. goodluck
Hi,
perhaps your primary Ab recognizes a protein present in milk? ;)
Check Ab info card for compatibility with milk.
Check milk composition (e.g. http://www.emeraldinsight.com/journals.htm?articleid=866460).
Try blocking in 2-3% BSA in TBS-Nonidet or PBS-Nonidet.
Of course perform proper membrane washes, as suggested by Bharat!
Best,
Lukasz
Hi,
I agree with Lukasz. Your primary may recognize something in the milk.
Also,I have had this happen with one of my antibodies and the solution that time was to block in 9% milk and to perform the last wash overnight in 4C (no rocking required during that last one). This resulted in a beautiful western.
Best of luck!
Gisela
Yes, last wash is the last one you would do (after the secondary). :)
Hi Mirza,
This is common problem everyone face in research field. I saw most useful replays above, i too suggest you few quick things..
Ab dilution things is differ from company to companies, so here i will go to use santacruz Ab's...
1. Increase your blocking time, use 5% milk in room temp for 3 to 4h or O/N in 4C with mild shacking...
2. 1Ab dilution you may go 1:1000 to 1:4000, don't worry about this step, keep it room temp shacking for 2 to 3h in 3 to 5% milk...
3. Wash you membrane 3 times in 15min intervals...
4. Go for 2Ab using 3 to 5% milk for 1 to 2h in room temp, use the dilution range is 1:10,000/ 12,000 also ok...
5. Wash you membrane 3 times in 15min intervals...
6. Add you mixture of chemiluminance in dark room, wait for 3min and go for develop your membrane..
This may help you,
Good Luck...
I agree with Aarti. Often more is not better. (I have experienced this in immunoprecipitations where I now immunoprecipitate for 20 minutes instead of overnight which used to be customary.) Although more is better in the wash steps in western blotting.
I also wanted to highlight another thing Aarti said: Carnation dry milk! I tried other brands when Carnation was out of production for a year or two and found that all other brands gave incredibly high background. I don't know what the difference is but it makes a world of a difference in western blotting.
Best,
Gisela
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