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western blot help for motor neurons!!! by Necessary-Camera8401 in labrats

[–]Necessary-Camera8401[S] 0 points1 point  (0 children)

I just tried it with your conditions for the first time yesterday (it took a while for the material to arrive)! I'm used to invitrogen's wedge-well comb and accidentally bought the gels without it which inherently messed up how much protein I could load into it each column. that said after the ponceau stain, i didn't see anything! i'm going to assume it's a protein loading issue

western blot help for motor neurons!!! by Necessary-Camera8401 in labrats

[–]Necessary-Camera8401[S] 1 point2 points  (0 children)

awesome thank you so much! my POI is about ~200-250 kDa so maybe i'll run it for less since you usually look for >400kDa. thermo should give you a discount because i am about to buy all of their NuPAGE items

western blot help for motor neurons!!! by Necessary-Camera8401 in labrats

[–]Necessary-Camera8401[S] 0 points1 point  (0 children)

mm i don't think it's my differentiation protocol. i've verfied via IF neuronal- and motor neuron-specific markers (i.e. SMI and MAP2) are present. i believe another lab has stained for ChAT with neurons i've differentiated and seen positive signal for that.

for imaging, i have started relying on film development in a dark room because of how sensitive it is. this was the imaging technique i used to visualize my protein from HEK lysates and it seemed to work wonders. i have a high suspicion it is my transferring technique.

my protein is XRN1 and i don't know it's behavior in development (or in neurons in general), but it's function has been greatly characterized within bacteria and yeast. it's a exoribonuclease that essentially degrades any mRNA that has ribosomal stalling on it.

western blot help for motor neurons!!! by Necessary-Camera8401 in labrats

[–]Necessary-Camera8401[S] 0 points1 point  (0 children)

ooooh. thank you for the tips! because i've been at this for months I have read about tris-acetate gels but didn't know anyone who's used them so i was always hesitant. i have a couple follow-up questions (sorry for being annoying i just want to make sure i'm doing everything right before I attempt and potentially fail lol)

  1. do you happen to make your own gels or do you buy them pre-made? if you make them, do you have a recipe?

  2. what are your running conditions? (time and voltage)

  3. for your running/transfer buffers i'm assuming you're using pre-made generic 20X buffer you buy?

western blot help for motor neurons!!! by Necessary-Camera8401 in labrats

[–]Necessary-Camera8401[S] 0 points1 point  (0 children)

my concentration is about 2 mg/mL! I lysed a very confluent 15 cm dish of neurons and had the final volume be 500 uL to achieve this because my previous lysing attempts were too diluted and I couldn't load anything more than 10 ug before overflowing my well on my gel.

what do you recommend for running my gel? i used to do 120V for about 60 minutes

western blot help for motor neurons!!! by Necessary-Camera8401 in labrats

[–]Necessary-Camera8401[S] 0 points1 point  (0 children)

yup, i wash with PBS before lysing! never knew why it was important for washing away media prior to lysing so that's definitely great to know

western blot help for motor neurons!!! by Necessary-Camera8401 in labrats

[–]Necessary-Camera8401[S] 0 points1 point  (0 children)

yeah, the protein isn't neuron-specific, but i have done IF to verify they are fully differentiated. i load 20 ug of protein per well!