The antibody (and exact protocol) that I'm using worked in the recent past, but now I get very high background, along with a "ladder" of negative ghost bands against the dark background. My protein of interest still faintly shows up as a positive (dark) band, despite all the non-specific ghost bands. But from my reading, ghost bands supposedly indicate the binding of too much secondary antibody. Does this imply that my primary antibody is somehow preventing binding of the secondary? By the way, the secondary is working fine with other primaries.
I've attached an image of the scanned film: lane 1=protein positive control, lane 2=knockout tissue, lane 3=wildtype tissue, lanes 3,4=experimentals.
1.Dilute the protein first. Dilution will help to lower the non specific binding of antibody
2. After dilution, spin the diluted protein homogenate at high speed for 10-15 min. Take the clear supernatant and load it on to the gel.
3. Try casting gels with new acramide, use new APS, TEMED for polymerization
4. Use new running buffer. Check for the transfer buffer (If possible make new ones) Are you using nitro cellulose or PVDF.
5. Do not reuse the primary antibody. Is your primary antibody raised in goat. If yes, use 5% BSA blocking and antibody dilution instead of 5% non fat milk powder.
Let me know if you have any questions and hope these points might solve your problem.
I sometimes see such ghost bands with a particular combination of primary and secondary antibodies. So, your secondary might work with other antibodies and also with the same antibody, albeit with a higher background.
If these are cell lysates you may try reducing the amount of lysate you load on to the gel. Alternatively, you can try a different secondary antibody like AP conjugated secondary or infra red dye conjugated secondary (I tried the IR dye conjugate and I obtained clean blots with the same primary).
Hi Brad, you can decrease your protein load a little as u ll see towards the bottom of the image there are white bands which show negative staining due to high amount of protein. Also the blot seems patchy, so u can change to 5% blocking also with NFDM or BSA. Also despite this if you see non specific bands, decrease your secondary antibody concentration. Hope it helps :)
It happened once for me. The problem came from my BSA which was contaminated. I came back to the use of dry milk the time to order new BSA...
For better WB. I suggest you to block with milk powder but does not work always.. some time 4-5%BSA is the best and give 10mins washes two times. dilute your 2antibody and after that give 3-4 good washes each of 10mins with enough buffer.
Additional suggestion, we have found that which company the milk powder comes from greatly increases background. We have found that using the brand Carnation milk powder gives us little-no background due to the milk blocking step.
Since your are working with tissue
1. blocking the blot overnight with 5% BSA in TBS+0.05% Tween -20 (TBST buffer) may reduce the background,
2. Dilute the primary antibody in TBST + 5% BSA, incubate, wash 4 to 5 times (each wash last 5 min).
3. Dilute your secondary antibody in the same buffer and after incubation give 4 to 5 good washes with sufficient amount of buffer.
As your samples are from tissue extracts this can make a difference from cells. If your tissues are from a mouse, for example, do not use mouse antibodies as you are likely to pick up non-specific mouse protein,such as IgG
Blocking for an hour should be fine. We use 5% marvel with 0.5% BSA added for tissue lysates.
Other factors are mainly using the correct antibody dilutions, which can easily be checked. I would run a gel with markers and a sample repeated across the gel, then cut the membrane to try different antibody dilutions for comparison. The background is high anyway so trying different blocking solutions may help as well.
Are detecting using fluorescent secondary? Could it be autofluorescence? Did you tried to make a membrane (or a piece of membrane) without the first antibody?
Hi everyone, Thanks for all the suggestions. To answer some questions, I block with 5% milk in TBS-T for 2 hrs before adding primary antibodies (also in milk/TBS-T). I'm using HRP-conjugated secondaries with ECL prime. These are mouse and rat samples, with anti-rabbit secondaries (anti-mouse secondaries, with a mouse primary do give plenty of extra bands for the mouse samples only, but this is not the problem here).
Diluting the primary antibody further seems to work - before I was using 1:1000, but now 1:10 000 or even 1:20 000 seems to work better. I had to buy a new tube of primary since I did the original tests, so I suspect that I got a different batch that is much more sensitive than the original.
Thanks for the help!
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