The Job Hunt Thread

[NomoreDramaQ1015]

Oh I don't think anyone who works in UE is personally out to get him. I just think the state of Nebraska is bleeping crazy.  We're now first in the country for the strictest rules regarding unemployment claims.   Florida is a distant second.

Governor Ricketts is the one who thinks all this will solve Nebraska's UE issues.   Personally I think it has more to with cows still outnumbering people in a lot of areas and a massive meth problem, not people losing out on job counseling sessions.

[muttleynfelix]

Jan 5, 2016 13:24:12 GMT -5 NomoreDramaQ1015 said:

Well if something doesn't happen on the UE front we are officially in the red.   A decision is going to have to be made soon regarding Abby's daycare b/c we can't afford it on my salary alone and there is not enough in savings to cover it, we ate up our savings when I was unemployed.  I can't ask them to take an IOU till the god damn unemployment office figures things out.

I am carrying around so much anger I could puke.   I am mad at DH's former employer, I am mad at DH, I am mad at the unemployment office and I am mad at the situation as a whole.  Just when I thought all this BS was going to be over and things would finally go our way this happens.  All b/c DH had to  make a mistake and then his boss went nuclear on him compared to the bitch who has gotten away with everything short of murder.

I don't know what to do about how I feel, I'm about two seconds away from crying at any moment.  DH is depressed enough as it is and my job is to play the supportive loving spouse so I don't dare say anything to him.   I wanted to have a tantrum over my Western Blot not working but since I work with people now I cannot resort to the type of language that would make my father blush as I used to in my previous job.

I want to punch someone/something but that's not a good way to handle things either.

You guys have been through a lot the last year. That is a lot of really stressful things to happen to you. Venting, counseling, etc and just having an outlet is good. Hell, I found just listening to my cousin talk was therapeutic even though I didn't want to talk about my problems, but just hanging out and laughing with someone else was really helpful.

[TheHaitian]

Just take 1 day at a time drama, fingers crossed!

Glad your husband will be able to get UE, that will be one less thing to worry about.

How long is UE again in your neck of the woods?

[wvugurl26]

Fingers crossed it works out drama. You've been through so much lately you are due for something nice.

[NomoreDramaQ1015]

When I was applying it said you could potenitally qualify for up to six months.   God I hope DH isn't unemployed that long.    .

DH hasn't filed in decades so he should get the max you can be granted and get the maximum duration of time.

It all depends on what his company says is the reason they justified firing him.  The UE guy thinks it will be hard for them to justify they needed to fire DH based on what DH said the reason for termination was.   The UE guy even said it made no sense they went with termination for something that small.

But who knows what they concocted while he was gone to cover their asses.  I am hoping the VP is more honest than that but I don't trust anyone in this situation as far as I can throw them.

In other news I flew solo yesterday for the first time humanizing pups.   Everything on the surface looked good and it seems pretty hard to screw up so hopefully I did a good job, won't know for another 8 weeks.

Still fighting with the Western Blots.   This anti-body came up positive for mouse as well, they want me to do it AGAIN to prove that it cross reacted.   The postdoc I am working with (he's a freaking genius when it comes to WB) can't figure out why they just don't go straight to RNA.   I said me technician, them PI I am just doing what I am told.  

My question is according to them Japan verified these mice via ELISA.   Why not get their ELISA protocol and have me replicate it to see if I get similar results?   That would make the most sense to me.

[The Walk of the Penguin Mich]

Jan 6, 2016 9:07:54 GMT -5 NomoreDramaQ1015 said:

Still fighting with the Western Blots.   This anti-body came up positive for mouse as well, they want me to do it AGAIN to prove that it cross reacted.   The postdoc I am working with (he's a freaking genius when it comes to WB) can't figure out why they just don't go straight to RNA.   I said me technician, them PI I am just doing what I am told.  

My question is according to them Japan verified these mice via ELISA.   Why not get their ELISA protocol and have me replicate it to see if I get similar results?   That would make the most sense to me.

Because Western Blot is much more specific as to which antigen the antibody responds to.  It tells you if the antibody is specific to only one protein, or multiple proteins.  Unless you have a pure protein (as determined by WB) to coat on your plates for ELISA, you don't know what your protein is positive to.

I've done a buttload of WB, hate doing them because they are so flaky.  

I'm guessing that you have run your secondary antibodies on the blot as a negative control.  It is not unheard of for those to cause problems too.

[NomoreDramaQ1015]

Well then my next question is if Japan didn't do a western blot then how do they know if they got human IL-34 or mouse?  I think I will keep that question to myself, I am afraid of the answer.

The mice are expressing both since it's a transgenetic mouse. My problem is all the antibodies on the market cross react b/c the majority of the time you don't give a shit.   I get the joy of dealing with a mouse expressing both.

 My life is being made much more complicated by the fact it expresses both and I have no way to tell if a mouse is a double positive or a heterozygote when I breed, all I know is the gene is present.

But they want me to breed them to homozygocity.  I told them honestly I doubt that will happen. I will try but since I can't control what I am breeding it's a crapshoot as to when/if I will get an all positive litter (which means I finally achieved the desired breeding scheme).



IMO it would have been better for this mice to have been designed as a knock in/out.   Then it would have been easy to get the desired genotype in just a couple of generations and I would be having a lot less trouble validating gene expression.

OR OR they could have sent some males along with those four females so I had the desired breeding scheme already in hand, but NO.  They only sent four females which had to be bred to wild type males to make babies, setting this whole clusterf*ck in motion.

[The Walk of the Penguin Mich]

Jan 6, 2016 12:56:13 GMT -5 NomoreDramaQ1015 said:

Well then my next question is if Japan didn't do a western blot then how do they know if they got human IL-34 or mouse?  I think I will keep that question to myself, I am afraid of the answer.

The mice are expressing both since it's a transgenetic mouse. My problem is all the antibodies on the market cross react b/c the majority of the time you don't give a shit.   I get the joy of dealing with a mouse expressing both.

 My life is being made much more complicated by the fact it expresses both and I have no way to tell if a mouse is a double positive or a heterozygote when I breed, all I know is the gene is present.

But they want me to breed them to homozygocity.  I told them honestly I doubt that will happen. I will try but since I can't control what I am breeding it's a crapshoot as to when/if I will get an all positive litter (which means I finally achieved the desired breeding scheme).



IMO it would have been better for this mice to have been designed as a knock in/out.   Then it would have been easy to get the desired genotype in just a couple of generations and I would be having a lot less trouble validating gene expression.

OR OR they could have sent some males along with those four females so I had the desired breeding scheme already in hand, but NO.  They only sent four females which had to be bred to wild type males to make babies, setting this whole clusterf*ck in motion.

Drama, I'm going to take this to PM and talk to you about this.  I have some ideas as to what's going on.

[myrrh]

Drama for what it's worth I am sorry about your DH's employment situation. Family owned companies can be ridiculously unfair to non-family members. I hope he finds something better soon. 

[NomoreDramaQ1015]

DH has an interview on Friday for Omaha Steaks.   He sent the application in less than 24 hours ago.  I take them calling back so soon as a good sign.

He's interested in seeing what they call the "cleanest plant in Omaha".   I said if you do find something that would show how good a QA you are!

[NancysSummerSip]

Wow, who knew all this was going on with so many!

I posted over on EE, so I will keep it brief. I was laid off yesterday, and my company is closing down for good next month.

I'm doing OK at the moment. Six weeks of severance pay, medical coverage for the next six weeks, and one job interview lined up for this week. And FWIW, I did see this coming. The company has been going nowhere for months. And I really hated my job. I figure yesterday was either the worst or best day of the rest of my life. I'm going to try to look at it as the best, because I get to start over.

I have savings, both local and in online account. House and cars are debt free, we have plenty of food in pantry and freezer and DH has his health care covered, along with his pensions and investments and Social Security. At least I do not have to worry about him as well as myself.

My resume was ready anyway, so even with the interview coming up, I am still looking and letting friends know my situation. I am also a freelance writer, so still working that angle to keep the income stream going there.

[shanendoah]

NomoreDramaQ1015 - I know he doesn't want to hear it, but wishing your DH good thoughts for Friday. I would also say them calling him back within 24 hours is a good sign.

NancysSummerSip - Sorry to hear about the layoff, but I agree you should think about yesterday as the best day of the rest of your life. You had been talking about trying to find a new job over on EE for years. And now, you'll no longer have to put up with the hag. So while it sucks to not leave on exactly your own timeline, I am thinking this is the kick you needed to the new path you've been wanting to take.

[NomoreDramaQ1015]

DH told me it was okay to wish him good luck for this job.  He wants this one.

Nancy I'd say it's the best day of your life.  No more Office Hag!

[NancysSummerSip]

The Hag was in the boss' office when I left. Of course she was pissing, moaning and crying about her financial situation. Her DH's salary cannot sustain them, not with their lifestyle. Designer dogs, a new and expensive truck they bought with pretty much nothing down, a house that's a shambles, plus their eating out lifestyle. She wouldn't use a coupon if God himself gave it to her.

As for me...my grocery list is ready for tomorrow, with coupons attached. It's pretty minimal, but that's fine. I am doing no driving today, saving it all for tomorrow and doing all the chores at once. At the moment, I will not change any of my account deductions: my retirement, new car, HSA and vacation/competition accounts will remain as they are. If I don't find work within three weeks, I will re-evaluate all that.

I am maintaining my workouts: they are monthly costs, but not huge ones, and I think they are necessary to maintain my sanity. I may have to cut back on some competitions, but I can deal with that.

My big worry is health insurance. There is no way I can go without it. DH is medicare, so that's no help. I will go COBRA for a short time if I need to, but likely look at the ACA as well. I have dental and long-term insurance on my own, and plan to keep them. I'm too old not to.

[NomoreDramaQ1015]

Her DH's salary cannot sustain them, not with their lifestyle. Designer dogs, a new and expensive truck they bought with pretty much nothing down, a house that's a shambles, plus their eating out lifestyle.

Well that makes me feel better about our situation. 

[mojothehelpermonkey]

Jan 6, 2016 13:52:54 GMT -5 The Walk of the Penguin Mich said:

Jan 6, 2016 12:56:13 GMT -5 NomoreDramaQ1015 said:

Well then my next question is if Japan didn't do a western blot then how do they know if they got human IL-34 or mouse?  I think I will keep that question to myself, I am afraid of the answer.

The mice are expressing both since it's a transgenetic mouse. My problem is all the antibodies on the market cross react b/c the majority of the time you don't give a shit.   I get the joy of dealing with a mouse expressing both.

 My life is being made much more complicated by the fact it expresses both and I have no way to tell if a mouse is a double positive or a heterozygote when I breed, all I know is the gene is present.

But they want me to breed them to homozygocity.  I told them honestly I doubt that will happen. I will try but since I can't control what I am breeding it's a crapshoot as to when/if I will get an all positive litter (which means I finally achieved the desired breeding scheme).



IMO it would have been better for this mice to have been designed as a knock in/out.   Then it would have been easy to get the desired genotype in just a couple of generations and I would be having a lot less trouble validating gene expression.

OR OR they could have sent some males along with those four females so I had the desired breeding scheme already in hand, but NO.  They only sent four females which had to be bred to wild type males to make babies, setting this whole clusterf*ck in motion.

Drama, I'm going to take this to PM and talk to you about this.  I have some ideas as to what's going on.

I was just going to chime in and say that ELISAs can be unreliable, especially if they used a commercially available kit and didn't run enough controls. For that reason, I could see them wanting to confirm the results with a western.
Anyway, it looks like Mich knows more about IL cross reactivity than I do. However, I do know from my tech support days that it is not something that you can necessarily trust most antibody vendors to accurately test for.

[NomoreDramaQ1015]

The antibody I am using now was verified in ELISA but it has never been tested in Western Blot.   So I am pretty much crossing my fingers here.

It's the best I could do, all the other commercially available IL-34 antibodies state upfront they cross react with mouse/rat.   Which isn't normally an issue b/c you're either working with human proteins or mouse proteins.  Not both in one animal.

If this WB doesn't work they said we'll move onto RNA work instead.   They wanted to do both but they said sometimes it doesn't always work out that way, they have another strain they haven't found an antibody for either.

[The Walk of the Penguin Mich]

I was just going to chime in and say that ELISAs can be unreliable, especially if they used a commercially available kit and didn't run enough controls. For that reason, I could see them wanting to confirm the results with a western.

Anyway, it looks like Mich knows more about IL cross reactivity than I do. However, I do know from my tech support days that it is not something that you can necessarily trust most antibody vendors to accurately test for.

An ELISA is only as good as the protein you coat the plate with.  If you coat the plate with a contaminated protein, then you increase the likelihood of a non-specific antibody response and you don't know what the response is to.  

I've spend my life developing ELISAs and WBs.  I've also argued with just about every biological supply company in the US with regards to their products, trouble shooting with them and telling them what is wrong with their product.  They will lie up one side and down the other that I am wrong - and then 6-12 months later, I'll get a letter from them saying "whoops, we goofed, here's a replacement product".  By then, I've usually moved on to another source.

Back in 1980s, we were looking at C-reactive protein in some of our inflammatory responses, so I developed an ELISA for that (I had to actually make both the antibodies and purify the proteins).  It was a phenomenally clean ELISA, and was waaaaaay (about 1000x) more sensitive and specific than the medical tests on the market at that time.  I am so kicking myself that I didn't figure out a way of marketing it.  I didn't realize that this marker would later become an important indicator for heart disease, and become a very well used today.

<sob>  I could be flying first class regularly.

[mojothehelpermonkey]

I just typed up another post basically saying that while I think there are some good antibodies and immunoassays on the market, you unfortunately cannot trust a lot of companies to be thorough and honest about confirming the reactivity of most RUO antibodies that are available. The internet ended up eating that post, and that is probably a good thing.
Anyway, I think it is a good idea that they want to use RNA to confirm the results. Also, as long as you are getting clear bands, it sounds like your blotting skills are fine. It is probably just an antibody fail.

[mojothehelpermonkey]

Jan 6, 2016 16:31:09 GMT -5 The Walk of the Penguin Mich said:

I was just going to chime in and say that ELISAs can be unreliable, especially if they used a commercially available kit and didn't run enough controls. For that reason, I could see them wanting to confirm the results with a western.

Anyway, it looks like Mich knows more about IL cross reactivity than I do. However, I do know from my tech support days that it is not something that you can necessarily trust most antibody vendors to accurately test for.

An ELISA is only as good as the protein you coat the plate with.  If you coat the plate with a contaminated protein, then you increase the likelihood of a non-specific antibody response and you don't know what the response is to.  

I've spend my life developing ELISAs and WBs.  I've also argued with just about every biological supply company in the US with regards to their products, trouble shooting with them and telling them what is wrong with their product.  They will lie up one side and down the other that I am wrong - and then 6-12 months later, I'll get a letter from them saying "whoops, we goofed, here's a replacement product".  By then, I've usually moved on to another source.

Back in 1980s, we were looking at C-reactive protein in some of our inflammatory responses, so I developed an ELISA for that (I had to actually make both the antibodies and purify the proteins).  It was a phenomenally clean ELISA, and was waaaaaay (about 1000x) more sensitive and specific than the medical tests on the market at that time.  I am so kicking myself that I didn't figure out a way of marketing it.  I didn't realize that this marker would later become an important indicator for heart disease, and become a very well used today.

<sob>  I could be flying first class regularly.

This was my last job. My hands were tied, and I consider one of my main accomplishments at that job to be getting them to remove a shitload of ELISA kits that were consistently generating false positives that someone who was not running proper controls would not notice.
I am still unemployed almost a year later. My main regret is taking that job in the first place.

[NomoreDramaQ1015]

I'm working with someone who is a super genius at Western Blot so it's not technique.   He helped me show that the last antibody was a fail.  He's pretty confident this is an antibody fail as well since it's never been confirmed to NOT cross react but the PIs want me to verify so that's what I am doing.  

I am so glad for his help, if he wasn't here I'd be hiding in the corner sobbing.    Protein work and immunology are not my strong suits.   Developmental biology/animal work/DNA/chemistry are my comfort zones.

They knew that when they hired me though.  My PI said don't worry about it by the time I leave here I'll be an expert in both.


I know you can't trust a lot of companies as far as you can throw them.  We had issues at Creighton all the time with Santa Cruz.  Unfortunately they had a monopoly over a particular antibody my boss wanted really bad so they had him by the balls.

[The Walk of the Penguin Mich]

Jan 6, 2016 16:53:35 GMT -5 mojothehelpermonkey said:

Jan 6, 2016 16:31:09 GMT -5 The Walk of the Penguin Mich said:

I was just going to chime in and say that ELISAs can be unreliable, especially if they used a commercially available kit and didn't run enough controls. For that reason, I could see them wanting to confirm the results with a western.

Anyway, it looks like Mich knows more about IL cross reactivity than I do. However, I do know from my tech support days that it is not something that you can necessarily trust most antibody vendors to accurately test for.

An ELISA is only as good as the protein you coat the plate with.  If you coat the plate with a contaminated protein, then you increase the likelihood of a non-specific antibody response and you don't know what the response is to.  

I've spend my life developing ELISAs and WBs.  I've also argued with just about every biological supply company in the US with regards to their products, trouble shooting with them and telling them what is wrong with their product.  They will lie up one side and down the other that I am wrong - and then 6-12 months later, I'll get a letter from them saying "whoops, we goofed, here's a replacement product".  By then, I've usually moved on to another source.

Back in 1980s, we were looking at C-reactive protein in some of our inflammatory responses, so I developed an ELISA for that (I had to actually make both the antibodies and purify the proteins).  It was a phenomenally clean ELISA, and was waaaaaay (about 1000x) more sensitive and specific than the medical tests on the market at that time.  I am so kicking myself that I didn't figure out a way of marketing it.  I didn't realize that this marker would later become an important indicator for heart disease, and become a very well used today.

<sob>  I could be flying first class regularly.

This was my last job. My hands were tied, and I consider one of my main accomplishments at that job to be getting them to remove a shitload of ELISA kits that were consistently generating false positives that someone who was not running proper controls would not notice.
I am still unemployed almost a year later. My main regret is taking that job in the first place.

I'm sorry about that.  

I had one batch of kits that I was running on our Luminex.  There was something funky with their streptavidin-PE.  I knew where the problem was and was able to circumvent it by filching from another source of SA-PE and replace what they supplied.  But that pissed me off, as I had to pay to replace the SA-PE in those other kits.  I called the company, explained exactly what I did and how I fixed it, and what the problem was.  We had about $8000 tied up in this batch of kits.

I was told that they didn't get this problem when they ran it, so it was obviously user error.  My gut is that someone misdiluted the SA-PE, and when I further diluted it (per the kit's instructions), it didn't give me a sufficient signal and the amplitude was nil.  Normal amplitude would be from 10-22,000 MFI and this would give me an amplitude of 10-200.  

I used the rest of the kits with my modification, got my data and moved on.  6 months later, they replaced $8000 in kits because they goofed because someone misdiluted the SA-PE that they supplied in the kits.  LOL.....I took that batch of kits and ran some preliminary data for another study.

[steph08]

Nancy - sorry for the layoff but here is hoping you find something awesome!

I had an in person interview today to follow up for a phone interview I had before Christmas. It seems pretty promising. Only problems are that I am 21 weeks pregnant and it is located in the big city that requires an hour and 15 minutes drive without traffic. They were open to working from home though so if I can get 2 or 3 days, and a good salary, I will probably take it. They don't know that I'm pregnant though. I was also the first to interview. So either they will judge everyone else against my awesomeness or they will forget about me by the end. Haha.

Otherwise, I will keep on keeping on at my job until after the baby is born.

[NancysSummerSip]

Jan 6, 2016 20:44:01 GMT -5 steph08 said:

Nancy - sorry for the layoff but here is hoping you find something awesome!

I had an in person interview today to follow up for a phone interview I had before Christmas. It seems pretty promising. Only problems are that I am 21 weeks pregnant and it is located in the big city that requires an hour and 15 minutes drive without traffic. They were open to working from home though so if I can get 2 or 3 days, and a good salary, I will probably take it. They don't know that I'm pregnant though. I was also the first to interview. So either they will judge everyone else against my awesomeness or they will forget about me by the end. Haha.

Otherwise, I will keep on keeping on at my job until after the baby is born.

Thanks Steph! I have an interview today, and make it my job to be looking for a job at least 6-7 hours a day, whether it is phone calls, research, finding more freelance or doing my regular freelance writing (to keep an income stream going). I will be fine, no matter what happens.

The job possibility today is much closer to home, no need for interstate driving. That would be nice. I would get back at least 45 minutes in my day! More training time, right?

[The Walk of the Penguin Mich]

Jan 6, 2016 15:03:59 GMT -5 NancysSummerSip said:

Wow, who knew all this was going on with so many!

I posted over on EE, so I will keep it brief. I was laid off yesterday, and my company is closing down for good next month.

I'm doing OK at the moment. Six weeks of severance pay, medical coverage for the next six weeks, and one job interview lined up for this week. And FWIW, I did see this coming. The company has been going nowhere for months. And I really hated my job. I figure yesterday was either the worst or best day of the rest of my life. I'm going to try to look at it as the best, because I get to start over.

I have savings, both local and in online account. House and cars are debt free, we have plenty of food in pantry and freezer and DH has his health care covered, along with his pensions and investments and Social Security. At least I do not have to worry about him as well as myself.

My resume was ready anyway, so even with the interview coming up, I am still looking and letting friends know my situation. I am also a freelance writer, so still working that angle to keep the income stream going there.

Crap!  I didn't see this!  I'm sorry, Nancy.

We're dealing with something similar in that SO's local office has closed.  He's on the payroll until the 18th of this month, and then will get 18 weeks of severance pay.  He did get one job offer, but is accepting a position as a consulting position right now.

[NancysSummerSip]

Aw, Mich....I did not know about that. That's a decent amount of severance - I got eight weeks' worth plus insurance. I did just get another positive response to a resume I sent out today, from a legal firm. Sounds interesting, it's work I know I can do, the hours are right and it's about 15 minutes from home. No benefits listed, so I responded and we will see what happens next.

[NomoreDramaQ1015]

Good luck Nancy and TD.

Moving into RNA tomorrow with the IL-34 mice, we're scraping protein work for the moment.   God I hope these mice are doing what Japan says they are.

I haven't been able to get any information beyond they did an ELISA.   After talking with the post doc, my experience with the antibodies and talking here I am having nightmares.  

You'd think they had to have done more than an ELISA to confirm these mice are what they say they are but nobody has told me otherwise.

This could get interesting.


The humanized mice are starting to come along nicely, hopefully I can get them into a cycle where I have a litter close to weaning while the next ones are being born.   It's been a bit of a rocky transition, I am glad I know what I am doing or it would have been a lot worse.

[TheHaitian]

I turn down the position with the other job, the most they could afford to pay is 65k.

As for my wife she is applying for a position with a Health insurance company as a project
Manager.

Good and bad:
- good because she will most likely pay more than she is making now.

- bad because she will most like pay more on her student loans than she is paying now. Basically will go from $468/month to ~$1,000/month.

I told her it is a tricky situation, her new job would need to net $500/month or $6,000/year net just to break even. We will see....

[Deleted]

Jan 7, 2016 13:24:04 GMT -5 TheHaitian said:

I turn down the position with the other job, the most they could afford to pay is 65k.

As for my wife she is applying for a position with a Health insurance company as a project
Manager.

Good and bad:
- good because she will most likely pay more than she is making now.

- bad because she will most like pay more on her student loans than she is paying now. Basically will go from $468/month to ~$1,000/month.

I told her it is a tricky situation, her new job would need to net $500/month or $6,000/year net just to break even. We will see....

Isn't your wife working on her PhD that the university is paying for?

[wvugurl26]

Jan 7, 2016 13:24:04 GMT -5 TheHaitian said:

I turn down the position with the other job, the most they could afford to pay is 65k.

As for my wife she is applying for a position with a Health insurance company as a project
Manager.

Good and bad:
- good because she will most likely pay more than she is making now.

- bad because she will most like pay more on her student loans than she is paying now. Basically will go from $468/month to ~$1,000/month.

I told her it is a tricky situation, her new job would need to net $500/month or $6,000/year net just to break even. We will see....

I know you don't want to lose money but I'd look at anything close to break even if she had upward potential with salary. Forgiveness is good but if she can transfer to private sector and make a lot more it could be better for you. Definitely a hard decision though.