My suggestion is that you change your primary antibody. I have also used anti-EGR1 antibodies in the past, and my experience with Santa Cruz is that the quality of antibodies varies hugely from one lot to the other. When you have so many bands on your blot, it means that the primary antibody is not specific at all (and you have verified that this does not come from your secondary). Either get Santa Cruz to replace the antibody, preferentially with one that has been purified (with the risk that the replacement will not be much better), or change company.

I have used anti-EGR1 antibodies from Cell signaling with good results in western blot. When you first use the new antibody, try something that you know induced EGR1 strongly, such as TPA (PMA). Compare with and without TPA, EGR1 should be seen induced by TPA, at 82 kDa. No other band should be seen, or very faint and far from this molecular weight so that there is no doubt about he EGR1 signal. Other than that, you will never be sure that you are really looking at EGR1 when you do these blots.

Good luck!

Hi Muntasir,

First of all, why do you spin after boiling? Upon boiling samples for WB you get condensation as a result of the heat and spinning the samples after boiling will dilute your samples and you will then disturb your protein concentration. Secondly, what percentage of gel are you running? What was your transferring conditions? What type of membrane are you using? I can't see bands at 82kD. For how long did you keep your samples at -20C? Storing lysates are best at -80C except if you add your sample buffer and boil then -20C is OK. Try to cut the membrane between 75-100 so you get less non-specific bands and I suggest 10% gel. Make sure that your actual primary does work because sometimes primary antibodies are just crab (try to find publications to maker sure the primary you're using does work).

HI Fawzi, Thanks for your reply. I am using AnyKD gel from biorad. The sample I ran was stored at -20C for only one day. I use nitrocellulose membrane 0.45um pore size and transfer at 110v for 70minutes at 4C. One of the postdocs in our lab is using the same antibody without any problem. So, do u reckon the problem is something else?

70 minutes (semi-dry) transfer (5 papers up and 5 papers down with the membrane and the gel in the middle?)

In addition to the comments and suggestion given by Fawzi I would also suggest you to run a good positive control for the protein.

NIH/3T3 whole cell lysate could be a choice per antibody datasheet.

http://datasheets.scbt.com/sc-110.pdf

Fawzi, I do wet transfer. So, one pad and one paper up and one pad and one paper down, gel and membrane in the middle.

Dear all,

Nothing wrong with the process of this WB for Egr-1. Just a bad antibody.

I did lots of Egr-1 WB. Please use Egr-1 rabbit monoclonal (1:1000, #4153, Cell Signaling Technology, Danvers, MA), which is not super sensitive, but very specific. Please see my paper titled as "Nuclear import of early growth response-1 involves importin-7 and the novel nuclear localization signal serine-proline-serine."

Good luck

I usually observe more number of bands when conc. of primary antibody is higher and reducing this sometimes work.. Or you may order a fresh lot of monoclonal antibody.

Best

Hi Muntasir,

Just 30 min blocking with 5% milk is not sufficient. I guess this is the major problem happening in your case of getting too many non-specific bands. Block the membrane for at least 2 hours at RT in 5% fat free milk.

Good Luck.

I am agree with the suggestion of Jinbiao Chen I think you should go for monoclonal antibody and please use 10% gel for better separation.

Do you have a control with reduced Egr-1 (siRNA knockdown or perhaps oxygenation level )? It would be nice to know whether one of your bands really is Egr-1. Secondly, Egr-1 does get phoshorylated (and you say that you are using phosphatase inhibitors), so the higher (apparent) molecular weight bands are potentially phosphorylated forms of the protein. Once you know which of your bands really is Egr-1 specific, you might try reducing the concentration of the primary antibody.

As suggested get a monoclonal antibody, block for 2 hrs at RT and keep +/- controls to check for specificity.

all the best

Playing with the blocking conditions may help. (BSA blocking instead of milk for instance) Or loading less total protein , 25µg sounds a lot. But most likely the sad truth is: more than half of the antibodys out there are just poor quality, despite being terribly expensive. Get another one. I usually triy at least 2 or 3 different providers. Look for papers with decent quality blots (Tip: use Google Image) and purchase the samne antibody. Also, adding more Tween can help. But in most cases its just a lousy antibody. Sad but true.

Most probably your primary antibody is not good. Ask the vendor has anyone got good result using this antibody? If they can not provide evidence to say this is a good antibody. you suppose to get refund or at least exchange.

Controls is a good point, how many bands do you expect? What do others see?Some proteins give many bands because of extensive posttranslational modification/splice variants. If you have an expression plasmid you may want to include a lane with extracts from transfected cells, I also like the in-vitro transcription/translation (TNT) system, dump in the plasmid,wait an hour, get the protein. its a good reference, especially hen degradation in cells can be an issue. I totally agree wit Meiyi, they should give you a refund. But in my experioence almost no company does, Santa Cruz for instance gives you another antibody for free. I almost think some companies have outsourced quality control to thir customers: "no one complains? Must be good." ;-)

Good luck!

Hi all...thank you for your comments. I tried serum stimulating the cells which suppose to upregulate egr-1 expression significantly and serum starved cells should give very faint bands. But in both cases, i am getting strong signal @Ian Robbins.

I havent tried blocking peptides yet @Evonne Chin-Smith

I am expecting a single band at around 82kda. A post doc in my lab working with the same antibody and same cell line and treatment is getting it working fine. She is getting a crisp band at aroun 80Kda. But, its not working for me. @Thomas

I am trying to use a different phosphatase inhibitor to see if that makes a difference or not...If you have any other suggestion, then please write.regards.

Do what the post doc is doing side-by-side. Why reinvent the wheel if the wheel is rolling past you?

All right,

sample preparation:

please pipetting 20 times with 200 tips (yellow tips) after harvesting cells with RIPA. Store samples at -80oC. Others are OK.

Lots of suggestions from others are excellent. You can follow them if you want.

Can you tell some key information about this WB:

1. Egr-1 Ab incubation: time and temperature (in 5% milk/TBST)

2. Increase milk from 5% to 10% in blocking solution for 1 hour at room temperature, with shaking, not too fast

3. how long did you expose your film or under a digital camera (this is not related with bands, but very helpful for me to give a suggestion)?

Regards

Tried doing tht..but its not working in my hand.@ Jamie

Dear Chen, thanks for the comments. I incubate in 5% skim milk/TBST solution overnight. I use auto exposure. but it takes around 120seconds for the LAS machine to expose it correctly.regards

Under such circumstances, distribute the cell lysate in different fractions (preferably by using a suitable ion exchange chromatography and eluting with different strengths of a electrolyte say like NaCl) and then run Western Blot. It should work fine, only it will involve some extra effort if you wish to put in (no short cuts please).

You mentioned---"I am expecting a single band at around 82kda. A post doc in my lab working with the same antibody and same cell line and treatment is getting it working fine. She is getting a crisp band at aroun 80Kda. But, its not working for me."

Unless I am missing something, it's confusing. Is she doing the same/similar experiment? If it works for her, it should work for you. Why don't ask her to follow you when you are doing the experiment?

All other comments here are very good.

Solutions suggested for you:

1. The easiest and fasted way for you is to get a new antibody from cell signaling tech as mentioned before.

2. If you have to use the current antibody, which is not easy for many people, you can put all of steps in detail that you did in here from harvesting cells). I can go through them for you. Those bands are actually often picked up by this Ab (if it is sc-189). The only thing you can do is to differentiate Egr-1 bands from others and actually it is quite challenge.

Regards

My suggestion is that you change your primary antibody. I have also used anti-EGR1 antibodies in the past, and my experience with Santa Cruz is that the quality of antibodies varies hugely from one lot to the other. When you have so many bands on your blot, it means that the primary antibody is not specific at all (and you have verified that this does not come from your secondary). Either get Santa Cruz to replace the antibody, preferentially with one that has been purified (with the risk that the replacement will not be much better), or change company.

I have used anti-EGR1 antibodies from Cell signaling with good results in western blot. When you first use the new antibody, try something that you know induced EGR1 strongly, such as TPA (PMA). Compare with and without TPA, EGR1 should be seen induced by TPA, at 82 kDa. No other band should be seen, or very faint and far from this molecular weight so that there is no doubt about he EGR1 signal. Other than that, you will never be sure that you are really looking at EGR1 when you do these blots.

Good luck!

I would add lamelli buffer to the cell lysate and boit for 10 min THEN store them at -20. if you were storing your cell lysate without adding lamelli buffer and boiling you will have a high chance of protein degradation even if you added protease inhibitors. I would also suggest to increase the blocking time to overnight, or change 5% milk to 4-6% BSA instead and block overnight at 4C.

hope this helps.

Dear all, thanks for your helpful comments. I am working on your comments and will update you as soon as I find the result.

Do I add BME with the lamelli buffer before boiling or BME should be added just prior loading? @Maitham

you should mix BME with lamelli together and then add them to you cell lysate immediately then boil and either stole at -20 pr load into the gel.

Hi,

This may not at all be a technical issue. From what I can see on your blot it looks beautiful. Have you considered that your protein may be post-translationally modified? It could be glycosylated for example, which would change the size quite a bit. Certain cell sources of protein may display just one band for a given protein while other sources display several. You could try deglycosylating your protein before running it in your western (there is N-linked and O-linked glycosylation; since your protein is an intracelluar protein I would bet on O-linked; Q-Bio has good kits for deglycosylation). There are also other modifications that can be present that change the sizes of proteins.

Good Luck!!

Gisela

Hi, First and most important use a different antibody. Also get a control sample if you can from the post doc in your lab and run it alongside your samples. see what happens

antonio

Please correct me if I am wrong here:

On your membrane (attached figure), you are running: Marker and lysate

Short exposure time

1. Marker

2. Lysate (low concentration)

3. Marker

4. Lysate (high concentration)

Long exposure time

1. Marker

2. Lysate (low concentration)

3. Marker

4. Lysate (high concentration)

If this is true, you may have a contamination problem.

I don't have experience with that particular protein but when I got so many non-specific/unintended bands few optimization helped me overcome the issue such as blocking with and preparing antibody dilution in BSA, further diluting the primary and increasing (if needed) secondary, increasing wash frequency 10 times, 5 minutes each instead of 5 times, 10 minutes each. Also, increase the strength of your wash buffer, decrease the exposure time of the x-ray film, use lower to high sensitivity ECL detection reagent. I know these are very basic things but as I said these often fixed my problems. Good luck!

Clearly the primary antibody is not very specific or you have massive post-translational modifications and degradation (not very likely). If you decide to keep working with this antibody you should run the gel longer and use about 10% PAGE. Right now it looks like a band runs very close to other bands making them hard to distinguish from each other. Then increase blocking to 10% milk or use BSA or horse serum. Think 1h blocking at RT is fine. Since playing around with antibody concentrations did not give much of an effect I think your primary antibody is not very good to start with - anyway to compensate for this I would use about 1:200 - 1:600 dilution of primary and 1:5000 - 1:10000 of secondary. Try diluting antibodies in 1% BSA in TBST. Of note: secondary anti goat antibodies always give many unspecific bands in my hands - better dilute it. Also include a positive control if you have one (or negative EGR1 knockdown... etc). Good luck!!

I think you have some excellent suggestions. I would definitely try another antibody. Good I would also suggest trying a different blocking agent (BSA is fine as long as it is high quality-Fraction V). There are also some commercial blocking buffers which work well in my hands. Superblock by Pierce (part of thermfisher sci) works well and has protocols for WB. I would also consider switching to adding Laemelli buffer and heating prior to running. Finally, you cannot go wrong if you have one extra flask of cells that you stimulate with phorbol esters and another you do not stimulate. Then lyse and prepare as you do for other cells and run on gel with your test samples. having a positive and negative control will help. Good luck and let us know how things turn out.

Dear Friends..blocking overnight and adding a new phosphatase inhibitor solved the problem and now i get a single band..THanks a lot for the helpful suggestions ..But now I got a new problem..my control cells are showing increased Egr-1 expression..almost 2 folds more than the treatment group.. But the control group should express very low Egr-1 protein. What can be the cause of this problem? I serum starve the cell overnight prior treatment, which makes the cells quiescent and should keep the egr1 level very low...any suggestion?

Not really an answer to the second problem but a contribution to the initial problem. I had similar problem with connexin CX43. when I blocked overnight in Pierce superblock and increased the temperature of boiling to 90 for ten minutes, I got a single and clear band. My multiple band was because of a post translational modification giving rise to phosphorylated form of my protein of interest.

Yes. With some antibodies its hard to overcome the issue. You can try changing the source like vendor or going from anti-goat to anti-rabbit or anti-mouse. Hope you found the solution. Also, not to overlook the chances of antibody being spiked with other primaries. Just a thought. Good luck!

If it's not a non-specific binding of antibody, I think that is glycosylation effect (PTM). Lectin blot would be a good solution.

Use another primary antibody

The problem could be caused by two reasons:

1- The antibody that you are using is polyclonal. That kind of antibody is less specific than the monoclonal type, which can cause cross reaction e a non-specific marking in the western blotting membrane.

2- Check the conditions (time of exposure and solution utilized) of the block of the membrane. If the block is not correct the membrane of western blotting will apear with a lot of other non-specific bands.

Dear Muntasir,

to figure out what is the "correct band" I would use protein extract from 293 HEK cells transfected with an expression vector for Egr1. If Egr1 is reasonable stable and not modified to a large extent you should expect one most prominent band with your primary antibody.

And of course: to use a second or third primary antibody raised against Egr1 would certainly also be helpful (but costs money!). And keep in mind all the other suggestions given by the colleagues above.

Agree with suggestion to change the primary antibody Please also check using the right concentration of protease inhibitor and do not forget postive and negative control in your western blot. When all reaction parameters are correctly covered and you still have non single band then possibility of protein glycosylation and degradation may be considered

If post translational modifications are suspected, changing from skimmed milk to BSA as a blocking agent is always a good first step

May be you can try with antibody from other company and other solution - try to include the positive control sample from other cells or tissue, not from your experiment - you can compare.

Try another primery antibody.

I recomend looking into Maggy's suggestion of looking into protease inhibitor concentration.

also please see http://www.cell.com/cms/attachment/573683/4258988/mmc1.pdf

[method section]

Hi Muntasir:

Some advises:

1. Use a monoclonal antibody recognizing a single epitope and discard with specificity or cross-reactivity H9c2 cells.

2. Block non-specific binding sites using same solution but longer, overnight or a minimum of 12 Hrs.

3. Make an assessment of their antigen detected by Western blot assay using the minimum amount of antigen with the appropriate dilution of primary antibody according to the controls, conjugate dilution adjustment until clear specificity, zero inespecificity.

4 Use the following controls: 1. No primary antibody, 2 No secondary antibody and 3 Without any of the above and the antigen should be in different concentrations until not reaction.

5. Solubilize its antigen, for example, Triton X-100 1%, then you could remove some bands and solve your band of interest in the case your antigen be soluble.

Probably is not good the primary antibody. Have you tried with an immuno-precipitation of the protein of interest before to start with the w. blot?

In this way you should reduce the nonspecific bands.

Hi, Munatasir,

First idea:

the most (boring, frustrating) reason for too many bands in a western is a lousy antibody sold by the company. Its terribly expensive, but its usually worthwhile to try 3 or 4 different ones from different providers.

My "Standard procedure": I usually start using "Google image"(!), enter the name of the protein and "western blot" as search terms. Typically one gets a nice spectrum of western blots of highly variable quality ;-) , I go for the ones NOT showing a small stripe of membrane only (can be hard, as the bad habit spreads more and more, space restrictions in journals) If then I see one that looks good, I go to the paper and check the source of antibody (hopefiully mentioned) for my first attempt.

Next suggestion:

Do you have something, you could use as a positive and negative control? You can see many bands all of which could even be perfectly specific if:

a.) your protein has splice isoforms or is

b.) posttranslatonally modified or is

c.) processed/cleaved posttranslationally.

All these would be physiological and might possibly even be important for your study.

How do blots from other people look like? But even there be careful: For instance, the PML protein (which is what I am working on) migrates as at least 9 isoforms in western blot (splicing, SUMO modification, all true and validated!). Still many papers show a single band, which always drives me crazy (ah well..... lets not go there.) ;-)

Ideally you will find a cell line (or tissue) which is negative (knockout? other species?), and another one which is positive. Alternatively you find a treatment which is known to upgregulate Egr1 or you overexpress it by transient transfection. then you get a feel for where it should migrate in your gel system. (different SDS Gel systems (Bis-Tris/Tris-Glycin and so forth) give migration patterns. Do all the controls any you find out whats truly going on. TrustNo1 but your own data. Do it right, have proper controls, and you'll know once and for good.

My way to lyse cells is directly in Laemmli buffer (adding Benzonase to get rid of genomic DNA), about 2 minutes digest at room temp. 5' boling, centrifuge, store -20°C, centrifuge again after thawiing, load&run. Do not run too concentrated though.

Good luck and please don't hesitate to ask again!!

Thomas :-)

Ok sorry, I saw you solved the multiband-problem in the meantime. So many comments that this is easy to miss. Concerning your "new problem": If cells behave diferent from whats expected, Either the old data are not reproducible (Yes, that-happens!!), or the cells have changed. This is actually rather common: I recommend to go back to an old vial frozen in N2, as cells in culture do change (Genetic drift) and that can mess up your results big time. Plus definitely check for Mycoplasma.

Anyway, good luck and keep "the crowd" posted on your progress!!!

Thomas :-)

change the blocking agent: use BSA instead of skimmed milk

Use monoclonal primary antibody (preferably from Novus biologicals or Cell signaling)

Do the appropriate 3 time washing of the membrane after the treatment with particular antibodies

I suggest you block for 30 mins after incubating with your primary antibody and then wash and add secondary antibody.