Hi Jennifer,

The successful transfer of the ladder does not always mean that you have successful transfer of your protein of interest. It is always useful to stain the membrane with Ponceau S or amido black (do not use Coomassie blue for this purpose, although you can use it to stain the gel post-transfer to see how much protein is left on the gel).

Large proteins, in particular, can be difficult to transfer from the gel to the membrane while small proteins can transfer through the membrane. Using Immobilon-PSQ PVDF which has a smaller pore size than the more widely used Immobilon-P can prevent the latter from occurring. PCNA is rather small, so I would stain the membrane to check that proteins in the 20-30 kDa size range are present; your ladder proteins are present in excess so you might not notice such a pass-through effect.

I hope those suggestions help.

Does anyone else use the same primary or secondary antibodies that you are trying?  When you say the secondary is 1:2000 relative to the primary antibody, how exactly are you doing the dilution?  Normally the dilutions are relative to the milk/PBST solution, not relative to the other antibody.  One change you could try is to use PBS (no T) for the antibodies and PBST for the washes.  You can also try directly spotting a small amount of primary onto a bit of membrane (non-blocked, it will bind really well) and use that as a positive control for your secondary. 

Amy,

There was a publication that used this specific antibody and Abcam also guarantees that it works in yeast.

Sorry that was confusing the way I wrote it. For the dilutions I meant 1:5000 primary and 1:5000 secondary for one trial and then I tried 1:1000 primary and 1:2000 secondary for the second trial. The dilution is relative to the milk/PBST solution

Is the Tween not needed for antibodies? I'm not knowledgeable about how the different reagents help make the Western work

i like the idea of spotting a small amount of primary onto a non-blocked membrane as a positive control! I will have to try that!

Thanks for the suggestions Mark and Thomas too!

I will try staining the membrane with Ponceau to see if there is protein and if there is, I will try probing with an antibody that should give signal

Fingers crossed!

Hi Jennifer,

The dilutions you used should be fine, thanks for the clarification.  The Tween is used to wash off non-specific binding and it is not necessary to include it in your antibody dilutions.  We used to use milk/PBS for blocking and antibody solutions, then included Tween in the wash solutions. 

Do you have any purified PCNA protein?  That would be the best positive control of all. Best of luck!

I don't know how abundant PCNA protein is.  If it's highly abundant, then 20 ug total protein might be enough, but you might want to consider loading more.

Oh.  A couple more things.  Remember that westerns are finicky; even people who run them all the time can sometimes run a western that is absolutely uninformative, and it is not always obvious where the problem lies.

Sometimes, while you are working out conditions, it can be useful to run a gel that is lane 1 marker lane 2 extract lane 3 marker lane 4 extract.  Once you've transferred them onto the membrane you can cut the membrane into two-lane strips so that you can run multiple conditions at the same time.  (Or if you do have PCNA to use as a positive control, you could use three lane strips.  Also, if you are going to use some other antibody as a loading control, and the protein is a different size from PCNA you can cut the strips using the marker as a measure of where the proteins would be so that you can work out conditions for different antibodies at the same time.)

Also, I've mentioned this one on a previous thread, and people were divided about how much difference they thought this would make, but the Bradford reaction does not always do well with detergent.  It is possible you may want to use another assay, such as the BCA assay to measure your protein, or use a sample buffer that does not contain SDS.

It's a good idea to use the positive control for PCNA (purified protein or overexpressed protein from the plasmid). This permit you to verify if your antibody is working and if the dilution is correct. if you don't have the positive control you should at least control the presence of any atandard like beta actin etc to verify if the transfer was succesful and if the proteins are non degradated. Do you use the protease inhibitor? You should add it in every sample.

I would try also to increment the exposure time.

Jenifer,

Anna brings up a good point. How were your cell lysates prepared? S. cerevisiae is chock full of proteases that can degrade your protein of interest and also phosphatases (that can interfere with your attempts to study post-translational modification of proteins). Cryodisruption is very useful for e.g. to study the phosphorylations on the yeast kinesins that I study.

You can definitely load more total protein as well. I've used 30-100 ug total protein (in crude lysate) as measured by A280.

Tween 20 has differing effects - in some cases it promotes specificity but in other cases it promotes non-specific binding. Some antibodies require it to detect their target epitope but most don't. Millipore also advises against including Tween 20 in the initial blocking buffer.

As it turns out, plain old water is, in fact, the most stringent wash solution for westerns. I would be happy to discuss this further when your westerns are giving you signal.

On a non-protein note, has anyone else been (successfully) using the same ECL reagents?  They can go bad. 

ECL Kits also can go wrong if they are old. You can easily prepare yourself with coumaric acid, Hydrogen peroxide and luminol.

Jennifer,

I looked up your antibody but was told "it isn't sold anymore". I was looking at the species of your antibody. Is it mouse or rabbit? This is a very common mistake to have a rabbit primaryand an "anti-mouse" secondary, or vice versa. Hope this helps!

Baylin

Well first load the sample normally as you do it usually then transfer normally also. 

The PVDF membrane should be activated for 1 min by shaking using methanol then shake for 30 seconds with distilled water.

Block the membrane in 5 percent fat free milk in PBS and 100 micro liters tween 20. and store at 4C overnight\

the next day put the membrane in blockign solution on a shaker for 2 hrs

then primary antibody 2 hrs diluted in blocking buffer and PBS on shaker

wash for 1 hr on the shaker

then put the 2nd antibody on shaker diluted with blocking and PBS

wash one hr on shaker

then develop

I am totally appreciate with Amira Fakih .You try the protocol will get good result.

You have indeed skipping the blocking and washing steps, you should try those: block 1 hour at RT or +4 C O/N with 5% skimmed milk in PBST before incubating with primary antibody. And wash 3 x 5 min with PBST between primary and secondary antibody and the same before incubation with ECL detection reagents. Double check that you are using the right secondary antibody for detection. It might also be that the primary antibody itself is not working and you will have to order a different one.