Hi, you could try to add some fresh mercaptoethanol to your second extract and boil and blot it again. If that doesn`t help you might have used different or otherwise transformed cells.

I've sometimes had issues with getting good transfer at the higher molecular weights. Have you confirmed you have a good transfer on both blots, either by Ponceau S or by a loading control near the expected molecular weight of BRCA1 (220 kDa)?

Also, it occurs to me that you didn't sonicate the second sample, which is understandable given it's low volume (30uL). I'm not sure if there is a commercial sonicator that could be used for so small a sample, as it would take a very fine probe and low power. However, the QIAshredder system and other similar systems may be able to achieve the same effect (shearing gDNA, etc) [https://www.qiagen.com/us/resources/faq?id=5fdaa264-271c-4371-9099-cdaeb7b7b304&lang=en]

Did you manage to detect a loading control (Beta Actin or tubulin, GAPDH, etc.) in both the old and new westerns? You might consider including a positive control along with new samples to ensure the lack of a result is not simply due to a technical problem with the blot (e.g. the antibody milk went sour, the transfer didn't work, you accidentally washed the membrane with 10xSDS, etc). Thankfully you already have such a positive control in the form of your old samples.

Thank you very much for your response Brian.

I actually did sonicate the second sample, despite of the small volume, which I thought might be the case I could not detect anything much - even though I don' t exactly understand why should sonication in small volume harm the extract...? I even could not detect the loading control and I am sure that this was not due to technical problems with transfer etc. since I nicely detected both BRCA1 and BRCA2, as well as the loading control using the old sample on the same membrane. Another thing that I am suspicious about was the Urea buffer which something could have gone wrong with. 

I've had problems sonicating low volumes using our lab's sonicator, technically a 'Sonic Dismembrator' (https://www.fishersci.com/shop/products/fisher-scientific-model-120-sonic-dismembrator-4/p-3974654_/). Even at low power it can aerate and 'foam' a sample easily. Once the sample is 'foamed' the risk of protein oxidation theoretically increases, and the sonication ceases to efficiently lyse cells and instead merely heats the sample. However, my lysis buffer is 2% SDS, which may be more prone to foaming than yours. If you included the old samples on the same blot this is a real mystery. Did you measure the protein concentration of the samples the same way (e.g. BCA)? Likewise, did you run a Ponceau S to confirm the protein concentration?  If not, a quick SDS-PAGE followed by Coomassie S may help figure out if it's merely a loading problem... if the samples aren't especially valuable.

Hi Anita

Couple of points/suggestions

1- your urea buffer is very unlikely to "go wrong"; something may be off with the new lysis buffer you made, although so long as you have sufficient urea your cells should come apart nicely.  Are you "cooking" after adjusting in SDS-PAGE sample buffer?  I assume you are...Not sure what you intend to do after but if you are simply concerned with westerns, it's OK to heat @ ~95 to ensure thorough denaturation.

2-Your problem could be with your protein load.  Make sure you probe for an internal control (Tubulin etc...or, better, an abundant nuclear protein of your choice e.g. PARP1) and/or stain with Ponceau.  In addition, include a positive control that used to work (if you have any left).

Good luck!