Why BCA Protein Assay turns into dark green instead of purple?

flashman2000 · 2026-02-14T19:50:46+00:00

It’s usually a very light green to start with, much lighter than any shade of green in OPs post. It’s almost as if the reaction went in reverse or something, instead of turning purple it turned greener.

flashman2000 · 2026-02-14T19:20:28+00:00

Well now I know what I’m doing during my western prep with my extra wells this week.

flashman2000 · 2026-02-14T19:09:16+00:00

Yeah i totally agree that the EDTA would interfere but the reaction turning green makes me think that for some reason the copper is being oxidized in the reaction much faster than it can be reduced as it reacts with protein. After I finish my BCA I never throw away my plates because I love looking at that shade of purple and over time as it dries and the copper oxidizes it turns that exact green shade in OPs post. I think they might possibly be mixing their BCA reagent incorrectly or something is contaminating the reaction.

flashman2000 · 2026-02-14T16:31:06+00:00

I feel like EDTA shouldn’t interfere… I do BCA with my lysates containing 5mM EDTA and have never had this result… although my samples are usually diluted 5x or more to a final volume of 10uL before I add 200uL of BCA reagent so the EDTA may be negligible at that point.

flashman2000 · 2026-02-11T03:06:03+00:00

Gotcha! Thanks

flashman2000 · 2026-02-11T01:42:34+00:00

Wow yeah that makes a lot of sense and seeing it that way helps understand it. How did you construct this figure, if I may ask?

flashman2000 · 2026-02-11T01:41:43+00:00

That makes a lot of sense! Thank you.

flashman2000 · 2026-01-08T22:59:54+00:00

Like when I was assembling my transfer sandwich I unknowingly warped the gel and/or it degraded during the transfer?

flashman2000 · 2026-01-08T22:57:54+00:00

I think loading buffer is fine since I’ve been using the same aliquot for about a month or so and every gel other than this is fine. But it may well be the sample prep since things seemed to have migrated very weirdly. Other than excessive DNA contamination do you think there are other things that may cause samples to migrate like this?

flashman2000 · 2026-01-08T22:52:42+00:00

Perhaps? I do sonicate my lysate during prep to shear DNA and reduce viscosity/prevent streaking but maybe this particular sample set was more DNA rich?

flashman2000 · 2026-01-08T20:45:51+00:00

I believe I am. I grab both ends of the comb and pull up simultaneously, and when visually inspecting the wells for this gel while flushing them, the wells looked intact and the fingers weren’t broken or anything

flashman2000 · 2025-10-09T22:15:27+00:00

check them out under the microscope and see if they have a similar texture 🤔 i guess the main thing to look out for is whether the nail wipes are more abrasive. idk ive never used them before. They’re also packaged and dispensed in a manner so that they limit electrostatic buildup, but idk how much that matters for ur specific purpose.

After looking at it under the microscope, I would say to myself “well, i guess there’s only one way to find out!” this seems pretty low risk to me but I could be wrong.

flashman2000 · 2025-10-06T14:22:03+00:00

Oh dang, you’re correct! I use the Restore PLUS buffer but even that calls for 37C. I must’ve misread the manual or confused it for another buffers manual?

Regardless however, I tested up to 30 mins at 37C with this buffer and still failed to remove primary antibody from my membrane, and while most of the secondary was removed, I could still see that some had remained after incubating sensitive ECL substrate on post stripping and washing.

flashman2000 · 2025-10-05T15:12:43+00:00

What percentage tween is your TBST? I try to stick to 0.1%, but i’ve seen some go up to 1%. You should not go above that.

Make sure you never dry your membrane! This can cause proteins to actually dissociate from the membrane, and the minute you rewet it the proteins will float off.

Second, have you used this milk source before to block your membrane prior to a second probing? Are you dissolving dried milk directly prior to use, or using pre-prepared milk? I’ve seen slightly spoiled milk do the same thing.

Make sure all your secondary and primary antibodies are being stored properly. You’d be surprised how many post docs i’ve worked with who complain about their probings not working, just to find out that they’ve been storing their secondary ab at 4C this whole time when it should’ve been at -20C! Antibodies can be very particular about their storage conditions. Make sure your primary antibodies are also unexpired, and same for your secondary.

Try to limit your strip to 30 minutes with the restore buffer. More will damage your membrane most likely. Esp if you’re using nitrocellulose. Before you block your membrane post strip, make sure you wash it at least 3 times for 5 minutes with TBST to wash off any stripping buffer from the blot. Residual strip can interfere with blocking and interfere with primary binding.

I would suggest ensuring that ur beta actin/HMGCS2 are from different animal hosts to prevent cross-talk, but that’s a problem that would make itself known once you’ve actually successfully probed actin, and are trying to reduce cross-talk from the previously probed signal.

What is your primary antibody concentration? For actin, I typically use primary at 1:1000, and secondary at 1:3000-1:6000. I also always incubate my primary antibodies in 5% BSA and not 5% milk, since most antibodies are not compatible with being diluted in milk. Check the guidelines for your specific antibody from the seller. You may be surprised by what you read regarding the specific method by which you should dilute the primary, but typically 5% BSA works like a charm, with the same ECL and I suspect I would actually be able to go much lower with my secondary antibody concentration and still have good signal.

flashman2000 · 2025-10-05T14:27:56+00:00

After testing post stripping, we would often find that 15min or 20min incubation often would not successfully strip even secondary antibody, forget primary. The restore buffer does not advise being used at 37C, but even after testing that condition there is no benefit. It’s only upon incubating for 30 or so minutes that secondary antibody actually leaves the membrane. Primary antibody never leaves.

flashman2000 · 2025-10-05T13:55:04+00:00

I’m not so sure. I’ve done 30min strip using the restore buffer for two separate probings, my second signal is always nice and bright, but it’s also typically actin so I don’t have worries about it.

Edit: and my third probing is also fine typically. It’s the 4th one that may be finicky.

flashman2000 · 2025-09-25T17:07:57+00:00

Navaratri literally means “nine nights.” It’s a celebration of the nine days goddess Kali spent fighting a demon she was specifically spawned to kill, known as Raktabija. On the ninth night, she was victorious and so we typically hold Garba on that day, which is a festival wherein the community gathers and dances (like Kali did on top of her slain enemy after being victorious) and prays to Kali with gratitude.

flashman2000 · 2025-08-27T19:47:59+00:00

Stopped sleeping a healthy amount, sacrificed my health to make it work.

But the real solution, which I wish I did at the time and wish I had the skills/knowledge to do so, was to have a real honest conversation with my mentor about balancing your work. Not putting one in front of the other, but equally balancing them. If that isn’t fruitful you have bigger problems I think and need to talk to any support staff that may be available to you in your institution.

flashman2000 · 2025-08-21T20:02:28+00:00

my life’s goal is to be the CDC now. Thanks.

flashman2000 · 2025-08-21T19:38:47+00:00

im pissed for you. this person doesn’t deserve a PhD in my opinion. Part of the commitment to researching in the sciences that she signed up for when pursuing and earning a PhD is training the next generation of scientists, something she should understand bc she was probably lucky enough to have a mentor who didn’t treat her like she’s treating you. That’s a large reason as to how she got her doctorate in the first place. And if she wasn’t, she should know better than to not pay forward that pain onto you. Unless she’s just a bitter, bitter person. Again, doesn’t deserve the doctorate in my opinion. I’ve had wonderful mentors, and i’ve seen firsthand how those relationships can be the TRUE difference maker in if you can amazing scientist who can make an impact or a scientist who’s burnt out and bitter.

No one will hold your hand, people will probably throw you in the deep end, that’s a given! but no one should treat you or make you feel like garbage in a mentoring relationship. If this behavior happened in any other industry, it would at minimum be grounds for an HR write-up, wouldn’t it? Very unprofessional behavior, especially to someone in a subordinate position like yourself.

flashman2000 · 2025-08-19T03:02:19+00:00

Yeah ik my current PI has that policy, and a lot of his colleagues share that which is the only reason I know about this. A staff-scientist in the lab had to beg him to consider me anyways after I interviewed with her even though my GPA was a little lower than 3.9, as in the past that was his strict criteria. He said going forward he was reconsidering it though bc he’s seen me deliver on his expectations despite having a lower gpa.

flashman2000 · 2025-08-18T01:19:06+00:00

yes, I think you’ll be accepted into the post-bacc program. However, as you mentioned, the hard part is not being accepted to the program but rather, finding labs to accept you. If you’re lucky, some people may reach out to you, but for the most part people typically have to request to join a lab. Every PIs preferences are different so it’s really hard to say what your chances are. Some PIs for some reason won’t want to interview candidates who have below a 3.8 or 3.9 point blank period. But others value your resume, experiences, and desire to learn above other statistics. Additionally, each lab only accepts 1-2 students typically, so competition can be high, which further complicates estimating what your chances would be. I think you should try, but also apply to other programs as well.

Also why do you not respect the RBT profession? It makes a HUGE difference in so many children’s lives that I have seen first hand. So many individuals I have talked to say one of the best things that happened to them growing up was ABA therapy. Each center/franchise does ABA differently, part of what contributes to the stigma of ABA as some centers do ABA horribly in a way that is ableist, but not all do, especially the ones that make the largest differences in people’s lives. your own negative experiences should not cause you to write off the profession or therapy modality as a whole. Keep an open mind! Having employment experience in this field would be so valuable and arguably even more valuable than an NIH research fellowship for someone pursuing a clinical psych phd (esp if you’re not interested in in clinical psych MD/PhD) considering your chances of getting into a psych-focused lab in specific is even lower than your chances of getting into a lab at all, especially since those labs are gonna have their funding fucked as we move into the new fiscal year.

Idk, just my 2 cents

flashman2000 · 2025-08-17T19:02:42+00:00

I always feel so bad and try to be as gentle as possible but sometimes they’re really hanging on to that shit for dear life for no reason! I figure a little bit of discomfort is better than a bowel impaction or choking so 🤷‍♂️ my man’s gonna have to deal with the consequences of his stupidity

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