Could be problems with the transfer to membrane. If the current flow is not uniform across the surface of the gel (e.g. poor electrical contact / bubbles in the centre), then it may transfer preferentially at the edges. Try doing a generic stain such as Ponceau red to check the transfer

Surely the problem is not with your quantification but most probably the washing after primary and secondary antibody incubation is not uniform.Generally the secondary antibody has a very high affinity and improper washing could give you these issues.I guess the problem would solve if you give an uniform washing.

How are you adding your ecl reagent to your membrane? if the ecl doesn't cover the membrane equally then the development onto film will be affected (less ecl = fainter bands).

Could be problems with the transfer to membrane. If the current flow is not uniform across the surface of the gel (e.g. poor electrical contact / bubbles in the centre), then it may transfer preferentially at the edges. Try doing a generic stain such as Ponceau red to check the transfer

The bands you get should be so called "white bands" or "antibands". The signals too strong until the HRP completely utilised the ECL substrate in the center of bands. Try to REDUCE your sample protein amount in each lane or the conc of secondary antibody.

For the non-uniform shift of pattern in protein bands from left to right, make sure the protein loaded is the same. Then make sure during the antibody incubation time, the skimmed milk with antibody MUST cover the whole surface. Or else the center part of the blot would not contact to the antibody, of course the bands will become fade and lighter.

Thanks

Are you using a rocking shaker when incubating 1ry and 2ndry? There maybe a problem with the acrylamide you're using.

I agree with Peter, I would also say the problem could with transfer to the membrane. If you are doing wet transfer, with time the cassettes become worn off. So more pressure is applied on the edges of the transfer sandwich. Thus you get better transfer on the edges.

I agree with Peter and Danijela, which transfer system do you use ?

Check if the chemiluminescence substrate is evenly distributed when applied for whatever amount of time you established. Placing the membrane in a not well-leveled surface could cause the substrate to accumulate in some zones and not in other. You can also spread the substrate gently with a pipette tip border during the incubation time to help with the distribution.

sounds like a transfer problem: just to add to suggestions above,

1. Make sure you have good contact of your membrane with the gel during transfer, it could be a simple problem that your transfer apparatus is bowed in the middle and therefore less efficient transfer is taking place. It should be tight across the whole gel. Sometimes old transfer apparatus get distorted over time.

2. It could be you due to incubation with antibodies and or substrate: make sure membrane is evenly covered all the way through

It may be due to transfer problem. to check the perfectness of transfer you can use ponceau stain and if it shows accurate transfer of the proteins, then you may go through antibody treatment. And in case of antibody treatment , make it confirm that antibody is spreading everywhere on the membrane equally.

I agree, I would check the protein concentration that you loaded that's appropriate for the gel system you are using, I would check how "nice" the gel looks before you transfer it, bubbles could affect too, and make sure that you have good membrane contact when transfering. Also shake the membrane covered with chemiluminescent evenly for the time assigned. Good luck!

I completely agree with Ian Wilkinson. There is probably a trouble with your transfer apparatus. But secondly, if you are using a 3D rocking shaker, make sur that the central area of your membrane is covered by you antibody solution as the edges, prefer a simple 2D rocking shaker

I would agree with everybody who has suggested ponceau staining just to confirm the transfer. If you have time try running two gels just loading all the wells with the same amount of one sample, so they should all be the same, coomassie stain one gel and transfer the other, stain with ponceau and do a blot with GAPDH or actin and see what happens. I doubt that the acrylamide is a problem, but the coomassie should let you know if there is. If it is a transfer problem then the ponceau should let you know if that is an issue. One comment about the transfer is that the pressure on the blot assembly should be even, and I have noticed that when using older equipment sometimes the grilles that hold the membranes, 3MM and sponges bows out and the pressure experienced by the gel/membrane sandwich varies. Counterintuitively sometimes more pressure often causes less efficient transfer. Try running 2 identical transfers at right angles to each other.

My guess though is that probably you have a problem with overlay of antibodies or ECL. In theory if you have insufficient volume of antibody using a 3d rocker or orbital shaker then the outside edges will be in contact with the solution more than the centre, so you could dilute you antibody(ies) a bit more and ensure that all your membrane is in contact with the solution at all times. Assuming that you are like the majority of the rest of the world and run mini gels, then you could try putting the membrane rolled up in a 50ml falcon tube and place that on a roller to ensure even coverage. Similarly with the application of ECL you may be trying to economise on ECL and are not putting enough on. In the long run it may be more cost effective in terms of time and money to put a bit more on to ensure an even coverage of membrane. The last thing that I would suggest is that if the gels run evenly and the transfer runs evenly then it is a problem with overlaying antibody or ECL. Good luck!

may be your protein not transferred properly or you not adding ECL properly to the membrane. stain your membrane with ponceau and look whether transfer was proper

Hi,

I have faced it sometimes.

1. Transfer differences. When you roll a glass rod or something to remove extra buffer from ur blotting sheets, see that you roll it even n carefully without any gap.

Hope you are taking a significantly larger blot than your membrane to have a better and even transfer. Do stain it with ponceau to see the differences atleast in your loading control. If there you see variable staining then of course there is inefficient transfer. Also cut the gel little extra if you have imp. samples at the end of your gel. As in take care that the ends are not the victim of bad transfer.

2. Yes Ab dilutions covering the memb. can be an issue while you rock your blot overnight. Try taking an appropriate size of a container if you use containers and pour the soln. Else, if you use platic covers,see that it rocks evenly all through out.

I would suggest, you someone else 's transfer apparatus for a day and eliminate the possibility no.1

Kalpita

I agree with the above suggestions that most likely the problem happened during blotting (transfer) or development (chemiluminescence).

To control the transfer efficiency, I normally stain the blot with Ponceau red after blotting. Because I always load equal amount of protein to each lane, I expect similar staining intensity of each lane. If not, the transfer efficiency is not uniform.

I've encountered such a problem when I switched to using an orbital (3D motion) shaker as Didier suggests, instead of the usual rocking (2D motion) platform. Nothing else was different and the transfer looked even by amido black staining. The problem with an orbital shaker is that if you are incubating your blot in a box, the antibody solution tends to swirl around the edges of the blot and does not cover the center well.

To solve the problem, use a seal-a-meal bag and a heat seal to hold your blot in close contact with the antibody solution at all times if you use an orbital shaker. If not, place your blot in a box on a 2D rocking platform.

I agree with others that it could be a transfer problem. I hesitate to think, though, that the rocker would effect the differential band staining. The antibodies should be equally distributed through the buffer. Antibodies would not "prefer" one side of a solution vs. another. Also I agree to check your gels. Make sure they are uniform and flat. If all else fails, I would suggest to only use the middle lanes for data analysis. In fact, I often only use the middle lanes (excluding the two outer left/right lanes) as the gels are typically more uniform in the middle. You can run the ladder in the middle lane if you prefer as well. The key to WB is what data to collect and how to appropriately analyze it. Be consistent and perform all your positive and negative controls and you should be good. Remember, most often WB is not quantitative, after all, ratios are usually used for analysis.

Kalkal, If it happens several times with different membrane may be you can try with another transfer buffer recipe or preparinf fresh one. Also, ECL substrate can be an issue in this, as suggested by others above. Try to put the substrate the most homogeneously possible, not only in distribution but in TIME. It depends on the type of ECL you are using, some substrates are designed for fast detection but also its signal fade is faster, so the order in which the lanes became in contact with the substrate can also be a matter. Western blotting tends to require a little of artistic skills to get a nice blot.

In my opinion, I 'm not really think is the transfer problem.

If so, you will get smearing protein pattern instead of bands.

The best way is showing us your WB pic.

Easier to identify the problem.

I agree with the others suggestions. When I had such problem, I was advised that it could've the substrate is being used up really quickly. So I added the ECL reagent and with less or no further incubation time, I was able to overcome this problem. I hope that helps.

It could be a problem with efficient transfer of your samples. You can stain the gel either with Ponceau Red or Coomassie blue to visualize s good transfer. If they are not good you will get that kind of uneven staining. Remember to have fair amount of solution with both primary and secondary antibody so they are fully covered. Otherwise part of the blot will be drying out and you will get uneven staining. Good luck.

I think this most frequently happens because of insufficient ECL or antibody covering the membrane and use of a orbital shaker Vs Rocking.

Use more ECL, dilute Antibody in a larger volume and use a Rocker for both the incubation. You can try this on your old membranes without the hassel of running new gels and transfer first.

This should most certainly solve your problem. If it does not then you can try ponceau staining and optimizing your transfer as suggested above.

I agree with the suggestions made above. In a situation like that I'd check the status of your transfer system for first. Try to borrow one that you know it works for doing again the same transfer from a colleague. If the problem persist, I would focus my attention on the way your going to distribute the ECL on the membrane.

Yes this is most likely a transfer problem. Trying to stain the gel with Ponceau should show if there is a transfer problem. In that case try using a a different transfer apparatus. Also when you add your ECL substrate try adding a little more and put your membrane in an opaque box on a shaker so that the substrate spreads over the membrane in an uniform manner!!

As I understand, there is no problem with transfer.

This is a problem with development - too strong signal will just burn it self.

You probably want to play with secondary abs or reduce the strength of ECL

Sometimes, the antibodies are not a specific as others and the detection is very low thus we need to compensate by longer exposure thus introducing more background. Also, the transfer could be a problem, either too long or too short. Best of luck!

Kalkal, when you are done transferring and before doing anything else, incubate your membrane with red protein stain "Ponceaue" for a few minutes and then wash it a few times with TBST (each time for a few second to get rid of excess dye) . You should be able to see the lanes and protein bands on the membrane as red bands. Pay attention to the pattern, you will know if you have less protein in the middle or not, If the intensity of the lanes are the same, then the problem is during 1) antibody incubation time (e.g. the middle section of the membrane is not covered enough by Ab solution while shaking) or 2) ECL incubation (the ECL is not covering the middle part of your membrane. One way to avoid these problem is to add a little bit more of both during incubation.

Hi Benjamin,

I suggest watching how the antibody solution washes across a blot in a box on an orbital shaker. Due to the "orbiting" movement and inertia, the bulk of the liquid will run across the edges of the blot. This is in contrast to a shaker that rocks only in one dimension like a see-saw or one that shakes in two dimensions on a flat plane. This is why even with a perfect transfer, if you use a 3D orbital shaker, fewer antibodies will have the chance to wash over the center of the blot as often as they do over the edges, and you end up with an underexposed center, from my personal experience. People I know who use orbital shakers routinely for their westerns will always seal their blot in a bag to ensure even distribution of antibody solution across their membranes.

Hi Kalkal,

It's been a few years since I have done WB but had some experience with "wet" vs. "semi-dry" transfer techniques. "Wet" transfers were troublesome and no matter how low the voltage or how long the transfer (and at 4degreesC), transfer was inconsistent between lanes. However, I remember the problem being that bands in more central lanes were brighter than outer lanes, opposite to what you are seeing. No problems with "semi-dry" transfers and this worked well with quantitative WB using Odyssey system.

Mark

In my opinion, It maybe related to current flow failure at running step or transferring step. Incubating with primary Ab or Secondary Ab undoubtedly effect on all lanes and would NOT restrict to some parts.

Best regard,

Hadi

It is 100% due to the ECL, locally used up before you put your film on.

Put the ECL solution onto clingfilm or Saranwrap and then incubate the membrane, and then stretch the clingfilm during the incubation, this generally draws the ECL solution across the whole surface of the membrane.

Also good point about the second antibody incubation, we used to incubate our membranes in a 50ml Falcon tube, with the antibodies and do the incubation on a roller rather than a shaking platform, especially if the volume is very small, this ensures all of the membrane surface gets covered.

Yes! It may happen with some too stronger signals including beta-actin, Histone H3, or GAPDH. You may dilute the antibody and short the exposure time.

Uneven bands may due to the transferring. Make sure that the gel is run at lower voltage with properly prepared transfer sandwich. Check the membrane with the red protein stain Ponceae. If you pass that step the problem with the antibody either primary or secondary or the buffer used. Use enough antibody and ECL medium to cover the membrane. I recommend to use shaker or rocker for antibody incubation and for washing steps.

Good luck!

see also: Amersham ECL Plus Western Blot Product Booklet

"(white) Negative bands generally accur when protein target is in exess and antibody concentrations are too high. The effect is caused by substrate depletion." This is especially true for GAPDH.

Just load less protein per lane.

If your problem has still not been solved:

1. Try conducting the whole experiment on a different apparatus both for running the gel as well as transfer so as to overcome any electrical bad contact.

2. Load rainbow markers in the 2 wells on the edges and follow the band separation separation, in fact always.

3.Reduce the protein loaded in the wells (use about 3 dilutions)

4. cut transfer membranes to size. ponceau check must for confirming even transfer.

5. if your primary antibody has been tried before with good results, then reduce the dilution (3 trial dilutions)

5.Try to put your membranes in a plastic and seal after adding antibodies(prim and sec).avoid bubbles for these can give rise to patchy ECL reaction later on.

6.Washing steps : 3-4 a must again. Adjust washing speed to rapid.

7. Secondary antibody even distribution as well conc are important for ECL reaction. (try 3 dilutions). Shake on a rocker.

8.Washes 3-4 must on rapid

9. Prepare fresh ECL reagent in dark before using and follow their instructions

10. Clean everything you use for ECL prior to reaction

11. Use new film (from another group) and see that it does not get exposed to even a bit of light. Cut and carry strips according to size before use and store in dark.

12. carry out ECL reaction on a clean plastic sheet placed on a flat surface as per protocol in dark. Pour it evenly with a clean pipette in drops. wipe the membranes on tissue paper ever so lightly using always gloves after reaction. Keep membrane on a clean plastic, double it up and secure sides with tape and place it for imaging for different periods of time.(select best). Solution for developing should be freshly made, put in clean dishes. the membranes should be held with a clean forceps and quickly developed by shaking (dont allow to stand ). dip in water and fix again with shaking. wash with water for some time. always use gloves during the whole procedure. if the ECL is exhausted due to excess sec antibody which is unevenly spread then reaction will be uneven.(dark on the outside and white on the inside).

Take the current membrane that you have and repeat the ECL addition process on a flat sheet of plastic or plastic wrap with continued pipetting of soln back to the center of the blot. I have seen similar fading due to curvature of the container bottom used in the process. If you still have issues then look at using a rocker during incubation/wash steps and if that does not do it, then the transfer process its self.

I concur with what others have said regarding overloaded protein and antibody concentrations being too high.

Try running a dilution series of a control sample, in duplicate or triplicate, on the same gel with rainbow loading markers between each replicate. Transfer as usual, then use a fresh razor blade to cut the membrane vertically between replicates (note: a 96-well plate lid makes for a great cutting guide!) Choose a primary antibody and make up several dilutions, depending on the number of replicate strips you have. Now use one strip per antibody concentration to complete the immunoblot procedure. This experiment you should give you a better idea of optimal protein loading and antibody concentration for future work.

Also, be sure you only have one membrane per container during the whole immunobloting procedure, from incubating antibodies to washing and development. Otherwise membranes tend to pile on top of each other during rotation leading to non-uniform banding patterns. Although it's tempting to double up membranes in a container to save antibody it's not worth the risk.

If limited antibody is a concern, try using smaller containers - ones that fit your membrane size. Try checking with your local craft or home improvement stores for inexpensive divided boxes like you'd use to store buttons or fishing tackle. Keep in mind you can always cut membranes to get as much data as possible from one gel. Just load a rainbow marker on your gel then cut your membrane horizontally as needed based on protein size. You can then test multiple antibodies on the same blot at the same time (assuming your proteins of interest are far enough apart based on kD).

Hope this helps and good luck!

Hey Kalkal.

In short... you might need thicker bread... Let me explain:

We had a similar problem with WB's at my previous lab. Band started to fade and eventually they turned into swirls in the centre of the blot. The more we repeated the assays... the worse it became. A colleague of mine (Michael Berry) eventually discovered that the simple problem was the transfer cartridges that were becoming warped from the heat in the transfer tank. Inside the "blot sandwich", the edges of the SDS gel were making contact with the transfer membrane but the centres was maybe 4mm apart. We popped them into some rapid boiling water till they softened, then clamped them onto a flat surface to straiten them, then covered them with ice. As a precautionary measure, we started using thicker sponges... or... as we called it... "thicker bread in the sandwich".

Months of our lives wasted ;-D

Hope this works and you can blot-a-lot!

Randall's advice is good, as a larger sandwich will provide a larger blotting buffer reservoir. The alternative is to use a tank blotter, in my hands (and with the 100-200 kDa membrane proteins I blot) they give superior results (more buffer, lower temperature...).

What type of membrane do you use? Again in my experience PVDF works better than nitrocellulose (higher affinity and capacity for proteins, greater mechanical strength). It is also a good idea to carefully wash the membrane after blotting to get rid of SDS, electrolysis products and the like. I routinely use a Ponceau staining/destaining process to check for errors during blotting like air bubbles. That seems to take care of interfering substances too.

I think you have problem with washing steps. If you use falcon for incubation could be made your membran curve. In the washing step this could be effect the quality of washing. I recommend you to check your washing buffer if it is OK, use more washing buffer.

Hello Kalkal:

I agree with most of the answers. There are few suggestions on this topic.

1. If it is pre-cast gel, check the date of expiry.

2. After placing everything for sandwich, run a 15ml tube across the sandich before clamping to make sure there is no air-bubble trapped.

3. Place a container full of ice inside the transfer tank so the chamber does not get  too hot.

4. Incubate the blots in a container that flat surface so the blots get covered with the buffer containing either primary and/or secondary antibody.

5. Do not let your blots dry completely during chemiluminescence procedure.

Hope it helps.