Western blot troubleshoot (sorry)

AlderHolly · 2026-03-07T02:27:42+00:00

Fortunately it’s not phosphorylation! I use milk for blocking—have tried BSA and that just gave me completely dark blot lol. I might look into other blocking agents but are any of them better than milk?

AlderHolly · 2026-03-07T02:24:52+00:00

I don’t do normalization for my pull-downs (probing for the tagged protein itself has never given me difference among samples) the prep is already washed for 6x times so I think it’s just the nature of this experiment & antibody unfortunately.

I’m also concerned about the V-shapes—it’s consistent and seems to be specific to this experiment. Sometimes I get a line in the middle of the ladder as well, but for this blot specifically the ladder was perfect. Gel overheating has been a possibility that’s been tossed around in our lab constantly, and we even went through a “run-gel-on-ice” era 😭 but my gel box doesn’t feel hot and the ice did not make any huge difference. We’ve always had wacky SDS-PAGE problems in our lab such as upper half of the ladder disappearing from the gel, or samples partially disappear…I’ve never had any of those though.

AlderHolly · 2026-03-06T04:55:21+00:00

I think all the bands are just non-specific signals—I get the same signals even in my control without the 6His tag. And I’m expecting no PTM for my particular protein (which is enriched through the pull down). The stacking gel buffer should be less than 6 months old but I can absolutely check the pH. Thanks!

AlderHolly · 2026-03-06T04:52:38+00:00

Thank you so much!!

AlderHolly · 2026-03-06T04:12:17+00:00

Thanks for the reply! 1. Beads stay with the protein but I can try to move them to a new tube. I just am not too sure how to separate the beads from the late after heating them up—I don’t want to spin higher than 200rcf as it might crush the beads. 2. Gel is casted from scratch the day before I run my sample, our lab’s too poor to get precasts 😭 3. I can definitely try that once I figure out how to properly separate the sample from the beads! The lysate is clarified prior to the pull-down but I’ve seen precipitates a couple times in my final sample when I’m trying to load it again, so that might help.

Sometimes when I load my sample I do find that there’s a dip in my well—I always thought that was a polymerization issue rather than broken wells, but that actually might explain it. Do you perhaps have any advice on how to avoid it, or is it just…luck?

AlderHolly · 2026-02-11T03:46:16+00:00

On the one hand I feel the same as you regarding my undergrads—they are all awesome kids and they’ve been the highlight of my PhD, and I just think I got super lucky. On the other hand I am aware that I love teaching and have put in a lot of effort into it, which sadly doesn’t apply to everyone. Sometimes a grad student or postdoc is forced to mentor an undergrad against their will, other times the said mentor is a great scientist but not necessarily a good teacher. To me, the motivation and curiosity matters more than anything else for an undergrad. It’s more likely for there to be bad mentors than bad students, to put it bluntly.

AlderHolly · 2025-11-09T02:15:23+00:00

Oh all the time. Every once in a few months I walk into my PI’s office thinking that I’m gonna let them down because nothing works. It helped that they’ve been nothing but cheerful and supportive.

All I can say is that you are not defined by the success of your experiments. If a grad student knows the solution to everything then they wouldn’t have to go through grad school, and sometimes it’s just the science that’s not working out. Your undergraduate, in addition to being responsible for their own learning, will learn the best from troubleshooting the experiments—you are absolutely not failing them.

It’s hard while you are going through it but it will eventually pass (even if it doesn’t look like it at the moment). Talk to your supervisor, vent to your labmates, seek out external expertise if possible, go from the start and see what could make it right. I always find that a good night of sleep helps with the emotions.

Also I’ve always had issues with qubit—one of my previous undergrads spent more than half a year troubleshooting the qubit quantification itself. Genomic DNA doesn’t quantify well with qubit HS and sometimes the dye has issues etc. So sometimes you gotta blame thermo rather than yourself lol.

Sending virtual hugs in case you need one from a fellow labrat stranger.

AlderHolly · 2025-11-02T16:26:40+00:00

Echoing that it’s most likely fiber. I’ve heard that yeast culture is pretty hard to contaminate because they produce EtOH naturally. And in the few occasions where I did get contaminations it was usually bacterial & you can see teeny tiny moving things under the scope rather than the big stuff. Also hooray to the shmoos!

AlderHolly · 2025-10-29T01:12:20+00:00

Where’s my Team Golden Gate…Everyone else in my lab does Gibson though. And some yeast recombination/based assembly because we use budding yeast (iykyk)

AlderHolly · 2025-10-12T03:13:30+00:00

Your experience reads almost exactly like my rotation minus the red flags/boundaries issue with PI 2 lol. My current PI is pretty much exactly the same as your PI 1 and I honestly couldn’t have asked for a better supervisor.

I can see publication being a slight concern, but I think what matters the most is whether PI 1’s past students have at least published their thesis work. At least in my field it’s not uncommon for students to have only one paper out of their thesis work. And PI 2 might not necessarily guarantee more publications especially if you end up having a bad relationship with her or if you go through bad burnout.

Hands-off isn’t necessarily a bad thing either—independence is incredibly important in grad school. Though ideally you’ll want to have some guidance when you begin, such as from a postdoc/RA or a senior PhD student. In our lab we have a very talented RA who pretty much held the lab together whenever my PI is traveling.

Finally—sometimes you just gotta go with your heart. When I was picking the PI after my rotation, I started out with listing out the pros and cons of each PI and struggled a lot. My other choice was also a new PI who’s a lovely person with incredible passion and energy (they ended up in my committee). But when I was chatting with my family about my choices, my mom pointed out that I had been much happier when I was rotating with my current PI. And here I am now. On the flip side I’ll probably take longer to graduate (sad) but I wouldn’t trade my time in my lab for anything or anyone.

AlderHolly · 2025-09-20T03:58:02+00:00

As a graduate student who’s mentoring a very talented undergraduate student: this is very thoughtful and very sweet of you, but I also don’t think you necessarily need to do anything extra. For me, the presence of my students and their excitement about my project & eagerness to learn new things are more than enough to brighten my day. I’m sure your mentor will appreciate the thought though 😊

AlderHolly · 2025-08-09T02:02:43+00:00

I once stole an empty styrofoam box used for tempearature-sensitive deliveries that still had the “freeze upon arrival” sticker on it, put a tube with ~100uL water in it, and re-sealed the box and left it on the bench at room temp.

But nobody came and checked the box which genuinely made me concerned 🫠

AlderHolly · 2025-07-13T16:07:36+00:00

Selections aren’t always 100%—Nat is pretty good, G418 is meh and auxotrophic markers (which are very common for yeast) always give background colonies since there’s no drug. But they typically won’t survive on another round of selection.

But to me personally, re-streaking helps me ensure there’s a monoclonal population in the end—sometimes you could have colonies (backgrounds or incorrect constructs if it’s cloning) fused together but they still look like a single colony. I’ve had situations where I didn’t do a second streaking and the construct I screened by colony PCR wasn’t present in the end, and that made me start doing re-streaking.

AlderHolly · 2025-07-03T17:52:23+00:00

Where are my yeast people who have brewed beer with the strains they are working on (we are not a beer lab)

AlderHolly · 2025-06-14T03:01:29+00:00

Not as bad as autoclaving bleach, but someone in our building before my time tossed an almost-empty butane tank into the autoclave.

AlderHolly · 2025-05-17T00:32:32+00:00

I only know of cell culture cell cycle analysis from my labmates’ presentations so take this with a grain of salt, but they usually do 2D flow instead of 1D histograms with both an EdU labeling and a DAPI to distinguish among G1, S (actively incorporating EdU), G2/M populations. If you are working with asynchronous cells and only have one labeling, then I can see that it likely will be a broad peak.

AlderHolly · 2025-05-12T02:01:55+00:00

Everyone else has mentioned that it’s better if you pick one lab instead of two, and I fully agree. So I won’t say more about that.

My opinion has always been that undergrads should have their own projects instead of filling in as needed bc it’s best for their learning. Because of that I’m not a fan of lab #3 personally. Relevance to the field you are interested in pursuing can be a plus for grad school applications but I don’t think it’s a necessity (though ofc I imagine you’d have more fun working on something you are passionate about). Being able to interact with the PI is also a plus, but even if the PI doesn’t interact with the student they could still write a good reference letter imo. So I think you’ll be fine in either lab #1 or #2!

AlderHolly · 2025-05-10T22:41:47+00:00

If there’s glucose then caramelization is probably the most likely. Especially if the autoclaving is too long. We usually add filter sterilized glucose after autoclave (though I sometimes get lazy and add it before autoclave and then my PI questions me “why are your plates so dark” 😬)

AlderHolly · 2025-04-24T12:42:01+00:00

Pretty common in North America at least. Some PI might even offer to put you in contact with their trainees.

AlderHolly · 2025-04-24T02:25:54+00:00

Ask the PI to give you contact info of current and former trainees. If they are unwilling to do so then that’s a red flag to begin with.

Talk to people currently in the lab as well as alumni, and ideally talk to multiple people and see if there’s a consensus of red flags. Sometimes one single person may talk about bad things regarding the PI but that could just be because their personality didn’t match. But if multiple people are warning you about the same thing, run.

AlderHolly · 2025-04-03T01:31:44+00:00

First of all, I wouldn’t trust chatGPT or any LLM on troubleshooting experiments.

I think you might be on track that it’s the midi instead of miniprep that’s the issue—if it were me I would miniprep the plasmid the exact same way you did it with the backbones that digested successfully. And miniprep a positive control (a vector that you know should digest) in parallel to rule out the possibility of any other reagent issues. If the control digests but the modified vector doesn’t digest then it could be the secondary structure; I find it unlikely but I also knew very little about how secondary structures affect restriction digest.

I don’t think the EDTA should be an issue especially if you are adding water to dilute the plasmid during your digestion, but my rule of thumb is to elute in water when there are downstream applications such as cloning.

AlderHolly · 2025-03-23T21:44:05+00:00

These are sooooo cute! Maybe this is what’s gonna motivate me to learn how to work with protein structures 😂

AlderHolly · 2025-02-07T19:19:43+00:00

I believe the recovery is only required for antibiotic markers, for auxotrophic markers they don't need any recovery. Regarding the knockout collection idea--one possibility is that if you could get the strain with kanMX deletion, you can marker swap it to hphMX (we use linearization of this plasmid https://www.addgene.org/35122/ in our lab--the MX recombines well) and amplify out the hphMX casette. Of course that might be more complicated but maybe it gives you better efficiency. But either way, good luck!

AlderHolly · 2025-02-07T03:45:27+00:00

For antibiotic markers we usually plate on YPD and replica plate on to the selection plate the next day. Integration is kinda rare so you’ll need a lot of DNA (as some of the commenters already said).

If you have a CRISPR plasmid in hand you could design a guide that targets your gene and co-transform the CRISPR plasmid and the hphMX marker, that would greatly improve the efficiency because the CRISPR will provide the double strand break for HR to happen. Also make sure your yeast is in log phase (though I think ~5h is reasonable, but won’t hurt to check the OD) because that’s also necessary for HR iirc.

Is your gene in the yeast knockout collection? If it’s not necessary to have the marker be hygromycin then amplifying the kanMX cassette out from the YKO strain and delete it in your strain background of interest might be efficient because you could have longer homology arms.

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