16 replies to this topic

[alex_osu3]

Hello evreyone,

I am doing the western blot with ECL method in mouse system. I transfer the proteins to PVDF membrane with an efficiency about 90-95% confirmed by both Commassie blue and Ponceau-S staining.  The secondary antibody is cojucated with AP. I get substrates from Molecular probe. With the help of AP the substrates can emit chemilluminescence and detected by the film from Amersham (exposure time either 60 or 20 seconds). The question is that I always get high background even with secondary antibody only. The concentrations for the 2nd antibody are 1:5,000, 1:10,000, and 1:15,000 in 1xTBS-T, respectively. I wonder if anyone out there use more diluted 2nd antibody and less exposure time or good method to reduce background?

Thanks in advance,

Alex

Edited by alex_osu3, 30 April 2004 - 01:39 PM.

[phdconsult]

PVDF membranes age -  either in the lab or at the warehouse where they are stored. Try nitrocellulose. I think old PVDF traps substrate too.

[alex_osu3]

But I just bought it from Bio-rad for 3 momths

Li

[phdconsult]

Your date of purchase of the product might be unrelated to the date of arrival of the sheets at the company warehouse- try Schleicher & Schuell or Amersham as alternatives.

[geness]

hello,alex_osu3

    In general, I use the blocking buffer to dilute the 2nd antibody , then  put  the sample at 37centigrade for 2h and the background maybe become better.You can have a try .

Good Luck!

[alex_osu3]

Hi,

   I still have problems with my WB.  I got some information from JacksonImmuno, which says that the commercial milk might have some contimination with bovine IgG and this bovine IgG can react with anti Goat IgG.  It means when you use 5% milk to do the blocking actually you block your membrane with Bovine IgG which can react with anti-goat IgG(your 2nd) and this is equal to block your membrane with 1st antibody(It is stupid but I don't think many of us think of it before doing the WB including myself ). So when you add the 2nd Ab it will bind to your first antibody and the bovine IgG. This is one of the major reason that causes high background in the ECL.  They suggest use the normal sera from the 2nd Ab to do the blocking.


Does anyone have this problem before or hear of it before?


Alex

Edited by alex_osu3, 30 April 2004 - 01:40 PM.

[geness]

Alex ,

  I am  sorry that I can not understand you completely. Can you explain the last sentence again, OK?

[alex_osu3]

Sorry in a hurry. What I try to say in the last sentense is that pre-block the membrane with normal sera from the SAME host species as the 2nd Ab. In another word if the 2nd Ab comes from rabbit then the normal sera from rabbit should be used to block the membrane.  If 2nd Ab is made from horse then normal horse sera should be used to block the membrane.   Or you can check their website at www.jacksonimmuno.com for more details.

Now I am waiting for the rabbit sera from Jacksonimmuno lab. Hopefully get some results on this weekend.

Alex

Edited by alex_osu3, 30 April 2004 - 01:41 PM.

[keithwu]

Does washing with 0.3% Tween 20 in TBS help?

[WesternBlotter]

ECL and film always give background problems irrespective which membrane is used.
The best way to over come this is to consider a gel doc system or even better a LI-COR odyssey which uses infrared labelled antibodies to image.  Infrared vs visible...infrared will always win with lower background since biomolecules don't fluoresce at this y.

[ramakn]

Hello Alex,
Regarding the dilutions of the secondary they can go as high as 100,000. The type of membrane used does not have any significant effect according to me for both nitrocellulose and PVDF work equally good for me. try using high salt buffer for your washes (containing 0.5M -1M NaCl). This helps is reducing unspecific binding. you could alos try using coupling solution for diluting your antibody. By coupling solution i mean diluting it with 1% sera.

Hopefully this helps you.

[flashboy]

i'm using a membrane based colorimetric detection method [sigma's proteoqwest system] and i get unbelievably high back ground, so high that i have to stop the reaction after 3 minutes of the whole membrane would be purple.

i block with their blocking col (3% BSA) overnight at 4 degrees and always use tween in my wash buffer.... any ideas? would marvel (non-milk fat) be better?

[boduolige]

I had the same problem with anti-goat HRP secondary antibody. I tried to optimize it by using different diluted anti-goat (from 1:100000 to 1:10000) and using 5% milk or 3% BSA without primary antibody. What I found is that 3% BSA yields much much cleaner background when I use 1:100000 diluted anti-goat. In addition, I always block the blot for 2 hrs only @RT which means that you have to get up early to run gel, transfer, block and blot:-(. but good thing is that you can get the result in one day.

Good luck

Edited by boduolige, 26 August 2004 - 07:33 AM.

[flashboy]

weird eh, i'm now using 5% non fat milk and am getting no background at all, even using my secondary at 1:1000 dilution!!!!! finally got it to work after 2 months of optimisation!

[sunmore]

The washings between each step should be appropriate. The background may even evolve from the Luminol in the aged ECL kit.