The spotty nature of the bands makes me suspect that there were a lot of air bubbles between the gel and the membrane during the transfer process.

I'm assuming that the ladder was the same in both "L" columns, therefore the poor quality of the left side might indicate the gel/membrane sandwich was looser on that end versus the clearer bands on the right.

I would try to use a roller or pipette to smooth out and squeeze out any air bubbles between the gel and membrane the next time you transfer.

Hi, Jagadish. I think you have also problem with run, in addition to transfer. 80 µg pre tarck for me is quite a lot, it look lke they do not concehtarte well in the stacking gel. Have you used same solutions for gel preparations? By the way, to get better separation, I would suggest to increase pH of the separation gel to 9.15, you will have more clear bands. Good luck!

I agree with Jason, the sandwich can be a problem, but in this case you would observe an ugly bands at least in few lines. The amount of 80ug is (as Taras indicated) too high (30ug is better). For me it seems at least one of the antibodies died ;(

How do you store/reuse the antibodies?

Dear Jagadish, I voted up your question in order to promote the well described blotting procedure with the picture. The picture tells more than any description. I 'would like to invite you to copy your picture to my collection, with the link to your question. Please find the link below:

https://www.researchgate.net/post/Can_you_show_the_picture_of_your_worst_Western_blot_and_explain_the_reasons_for_this_disaster_please

Thanks guys for the inputs. I had used the roller to remove air bubbles from the sandwich.

The gels were precast and I had used the same buffers as before, it had worked but did not work the second time.

I store the antibodies in -20C. Primary antibody incubated overnight at 4C, secondary antibody incubated at room temp for 1hr and protected against light.

How do I prevent antibody from getting destroyed?

Hi, Jagadish, Store antibody in small aliquots, do not frieze-defreese more than 3 times. Check you membrane with Ponceau after transfer to visualize protein on membrane. Put membrane with protein side up during incubation with antibody. 4°C overnight for primary antibody is OK, do not agitate membrane during this period. You can try to stain membrane (which you shown on the picture) again with Ponceau and you will see transfer efficiency. Once you have a Ponceau stain, you can come to clear conclusion what is going on with current experiment!

Good luck!

Dear Jagadish, please follow the advices of Taras, for me it seems you did everything well... This is the hardest situation to advice, since we start talking about the moon phase impact;) My two minor points are:

- run again the samples you were successful,

- check if there is in antibody datasheet "don't freeze". If there is I wouldn't panic, since I always freeze Santa-Cruz antibodies. In the datasheet there is always don't freeze, newer affected my antibodies. I don't know their trick, but the antibodies they sell are useless at the day of experience when stored at 4 degrees. Keep fingers cross!

Hi, Jagadish,

jsut stain your current membarne with Ponceau, and, if you will see a good protein bands, re-use same membrane with antibody again (after stripping). In the case if you will not see good protein band, re-run protein once more time, but the gel with pH 9,1 and not more than 30 mkg protein pre track, By the way, what was your track size? Good luck!

Thanks. I do not know the track size, but the gel is from Precise Tris-HEPES Gels, 10 x 8.5cm. Specifications of Precise Protein Gels (Tris-HEPES Gels)

Cassette dimensions: 10cm x 8.5cm x 4.5mm

Gel dimensions: 8cm x 5.8cm x 1mm

Storage conditions: up to 1 year at 4°C

Stacking gel: 4% polyacrylamide

Buffer system in gel: Tris-HCl, pH 7

SDS content: none

Required running buffer: Tris-HEPES-SDS (Part No. 28398)

Recommended sample buffer: Tris-HCl-SDS (any type)

Hi, Jagadish, thanks for the information. Size of track is appr. 5-6 mm, it is not optimal for 80 µg protein. Have you stained your "old" membrane (one you put in the picture) with PONCENAU? You will have immediate answer on many questions!

As usually in agreement with professor Taras Pasternak comments above;)

Hi, Jagadish,

by the way, here is an interesting points about WB: http://www.labtimes.org/labtimes/trick/tricks/2013_05.lasso

Hi,

To me this looks like a transfer problem, firstly I can see air bubbles, so be careful when you assemble your sandwich that you roll out all the air bubbles. Secondly I think that your sandwich is getting too hot! run it for longer at a lower current. I also prefer to run wet transfers as this happens less often and you can be in control of the temperature more readily bu running in a cold room or using pre-chilled buffers. Also stain your membrane with ponceau red and you gel with coomassie so you can determine the efficiency of transfer and whether you have air bubbles trapped in the sandwich

good luck

Everyone has brought up great points, I'd like to add one more: check the expiration date on your pre-cast gels.

I've never used Precise gels, but I know Bio-Rad's pre-cast gels degrade within a few months after their expiration date, and this can cause problems such as bands running unevenly, disintegration/brittleness of the gel after running, uneven transfer to the membrane, etc.

@Jagadish:

What samples did you use in the trial run? I guess it was not rat lung homogenate.. Also, are the primary antibodies same in both blots. Even same species???? As in anti-human SpA vs anti-rat SpA??

Here is what I see:

1. You are probing with two SECONDARY antibodies. One is red for the marker and the other is green for your primary antibody. Red+greeN=yellow !

2. In your test run, your primary or its secondary also reacted with the marker so when you detect your marker, you see Red+Green = Yellow for some of the bands in the marker. But you saw nice green bands specific for the primary. How do I guess it is primary that is cross-reacting .... read on !

3. In the lung homogenate blot everything is mostly red. So your primary is not behaving the same as it did in the test run. This can mean you (a). Forgot to add primary (b). The primary Ab has changed - even changing human-specific SpA to rat -specific SpA can make a big difference. (c). the dilution of primary ab is much less.

If the green secondary was causing background then you would have seen yellow bands for the marker also in the lung homogenate blot. So I a guess the marker-cross reactivity in test blot is from the primary.

If the secondary antibodies in both blots are exactly identical - no change in species or anti-rat vs anti-rabbit or any switch like that - then if the secondary green cross-reacted with the marker you should have seen yellow marker bands. AGAIN, if and only if the secondary antibodies in both blots are exactly identical. You see very weak yellow bands and an overall green cast on the blot, so you definitely added green secondary antibody.

This leaves the primary:

A. The primary Abs are not the same - I mean anti-rat SpA vs anti-human SpA or case like that.

B. Test run was on different cell type and not same rat lung homogenate.

C. IF everything is exactly identical between blots,, you forgot primary in rat lung blot or it did not work (no yellow marker bands).

I will let you fill in the required info !! Let me know !!

By the way, you have a lot of particulate material in you lysates. They are showing up in the lanes. So (a). 80 ug of protein is toot much or (b). I think you have lots of debris and DNA in the sample (even small fragmented pieces from sonication). If you centrifuged after sonication, then debris is ruled out. Then sonicate longer, and centrifuge sample AFTER BOILING again, before loading.

Hi Prem..

I admire your thought process. Your explanation enlightens me on some of the basics of the fluorescence.

To answer your questions-

1. The test sample had the same rat lung homogenate as the second one.

2. Its identical primary antibody which I used and its rat specific.

3. I clearly remember adding primary antibody.

4. Its identical secondary antibody.

5. I sonicated for couple of minutes and centrifuged for 10 mins at 4C

6. I centrifuged again after boiling.

Today I did ponceau stain on the membrane as suggested by Andrew.

I have attached. You can see it.

After looking at it I feel there might have been issue with the transfer. I might not have saturated the filter paper, could have heated and caused damage. But I still could not explain how only the samples got affected even though I see the ladder transferred.

Hi, Jagadish,

yes, it is a transfer and separation problems. It is also clear why you see relatively good markers: you load 80 µg to each track, and this is too much for good separation and transfer. In the track with marker you have much less proteins! Good luck!

By the way, I noticed you submitted your blot to Jan's "Art Gallery" !!!! Good !

It is not just a bad transfer. There is protein precipitation, because the Ponceau stain clearly shows that when compared to the marker, the effect is more than just transfer. The gel has "crashed". It could be seen in the red fluorescence also and was not a good sign.

1. Like Taras and me said before - Overloading and/or protein precipitation. I prefer a mixture of the problems as the answer, since 80 ug should at least run IN the well, especially since it is a 1.0 mm gel. It is on the high side, but will not "crash" through the gel. It will smear more likely. But still stay below 50 ug.

2. I now am reminded of this pattern very much of what happened to me. The protein ran in-between the glass plate and the gel. This can occur for many reasons, but one symptom is that the loading dye will run as a broad line and not a sharp one. This will cause the protein to precipitate. Remove combs from precast carefully.

3. A final thought is DNA interference but your test gel looked OK - this where the sonication came in and the centrifugation before loading.

But I still maintain the absence of yellow compared to the test run in the markers is also something to watch out for from the primary antibody. This cannot be explained by overloading of markers. Just double check you add the right amount. Marker cross-reactivity is common for all manufacturers with many antibodies.

Let me know how the next gel turns out !!

Good luck !

PS: Look at Figure 6 in the attached .pdf from Licor the makers of the Odyssey system. Nice example of cross-reactivity.

Here is Licor attachment:

Thanks for the input. I ran more samples (same rat lung homogenates). This time taking care to remove air bubbles, made sure to wet the filter paper well in the transfer process. The transfer went well. I could see some cross reactivity, but my main concern is I don't see the band at 40kdalton, Instead I see the band well at 10-15k dalton. I have attached the single and dual channel images.

Both channels

Channel 700 only

Channel 800

Hi, Jagadish, at least now you have a prefect run and prefect transfer! What are the 15 kDa band? Is it not-specific binding? Have you frezze-de-freeze your extract many times? May be your protein was degradated though time? Do you have protease inhibitors in the buffer?

If you look closely on the previous test run, you can see the same 15kDa band in all of it. The samples are new, only once I have thawed. I have PMSF as protease inhibitor.. Still its a mystery to me..

Hi, Jagadish, yes I have seen 15 kDa in the first Western, of course. Probably, you need top have in future some positive control, ea, well know antibody whcih is work well always in all of the cells.. In this case you can cut part of the membrane after transfer and incubate separately with the positive control. Anyway, you ,may need such a control as a control for protein loading amount.,

I agree.

This is most likely complete degradation of your protein. Very typical to see nice reactivity at the dye front !

Re-Probe the same blot for b-actin or some such loading control.

BUT before that, do you have other proteins that your lab uses routinely - like using "everyday" - and are sure of the results. REPROBE the SAME blot with this antibody. You need to get some info back about this particular expt rather than try stuff blindly. Also, since you do not have bands above 15 KDa, you need not strip too much, since other proteins IF INTACT, will come above this.

1. By using a known protein - other than loading controls - you can check if the whole expt is OK and the problem is with only your SpA protein, and also if the degradation is with your protein alone. The loading proteins usually do not degrade easily, unless there is wholesale degradation. The idea is to see if there is complete sample protease degradation or just some proteins.

2. If not possible, then use a protein like b-actin, tubulin, to reprobe and see at least if the band can be seen at the right MW, then you know the blot worked and your protein is the problem You can then concentrate on your protein.

3. If the reprobe works, your gel and blotting are fine and then you are confident protein is degrading. This can happen in many ways. BUT especially since you are sonicating, sample heating up is a problem. Do sonication in short bursts only. 20 secs bursts , then keep on ice for 2 mins to completely cool back the sample. Do about 3 rounds of this. This should give you more than enough protein to run the gel.

4. PMSF alone may not be enough. You should probably add Leupeptin, Pepstatin, Aprotinin at least. OR you can get the protease cocktail from Sigma -use it at 5x more than suggested. Generally, the higher concentration is needed in my experience for sure inhibition. Pierce cocktail is also good (at 5x conc).

Good luck !

I stripped for 20 mins and reprobed with Actin, I see good band for actin. Now I think the Surfactant A antibody must have died or the Surfactant protein might be sensitive and degrades than other proteins. I did not image after stripping (before reprobing), the residual band at 15kDa might be from the previous probing..

Hi, Jagadish, this is very good, at least you have now a good run, good transfer, good loading control and good labelling. So, be care about protease cocktail inhibitors and primary antibody. It is well known that some protein have a high probability of degradation, but actin is much stable.. Good luck!

Yup. You are set. You have to watch out for degradation at sonication step...keep everything cool and do it in 20s bursts. Frankly, a single 20s burst is more than enough to mash the tissue. Try this first to see if it improves protein recovery.

And add protease inhibitors. The sigma or pierce suggestrd earlier and at 5x more conc than suggested.

You sre almost there!

Some more points:

I will come back to the red+green=yellow. The absence of the marker cross-reactivity suggests that the primary antibody has a problem. This cross-reactivity is a very 'cheating' but sure way to follow blot progress. ;-). It has never "lied" to me !! I have used it in cases where I get cross-reactivity but no band as a good indicator that my original sample had a problem. In your case, I am leaning still towards using a fresh primary. BUT, since your test blot was not so far ago and I do not know the history of your antibody I cannot bet on it.

IF and only IF you have a red color secondary you can use with SpA... Your green channel is now occupied by actin, since it overlaps with your SpA MW. You could then re-probe SpA in red channel with a new SpA antibody and check this out.

I have my quirks about Westerns, I know !!