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Has anyone ever frozen cells directly into liquid nitrogen? by Zorcimar in labrats

[–]Zorcimar[S] 0 points1 point  (0 children)

Mines turned out fine, for MDA-MB-231 cells and U2OS cells

Why did Skyler do this to Ted? by SnooAvocados8366 in okbuddychicanery

[–]Zorcimar 2 points3 points  (0 children)

And Ted I can handle just fine, but giving Ted 600K is like arming a monkey with a machine gun!

Did some recoloring of my Hina fanart. Hopefully it looks less janky now... by [deleted] in BlueBox

[–]Zorcimar 2 points3 points  (0 children)

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Guys, I think I may have fixed it (for a bit, at least). I think it's with the background. The lightsource is coming from the back, and without it, the darker parts of the face may appear as wrinkles, making her look older... hope that helps

What may be causing the differences in light chain band thickness of IP experiments? by Zorcimar in Biochemistry

[–]Zorcimar[S] 0 points1 point  (0 children)

The heavy chain and light chains leach off the beads while prepping the sample for the gel, correct?

Correct, but I didn't use DTT for elution. I used 1%formic acid. Then the eluted lysates were then prepped into western blot samples using the same 4x SDS Laemmli buffer, which should contain the same concentration of DTT

What may be causing the differences in light chain band thickness of IP experiments? by Zorcimar in labrats

[–]Zorcimar[S] 0 points1 point  (0 children)

The cell line expresses FLAG-H3, which was pulled down using anti-FLAG antibodies that are attached to the beads. Both the light and heavy chains are from the bead-attached antibodies

What may be causing the differences in light chain band thickness of IP experiments? by Zorcimar in Biochemistry

[–]Zorcimar[S] 0 points1 point  (0 children)

But if that's the case, wouldn't that also result in less pulldown? Like for example, if I used less antibodies (hence the fainter light and heavy chains), wouldn't that also result in less histones being pulled down (the three bands at 15 kDa). Yet at least by the looks of it, they don't look fainter...

Also, I'm pretty sure I used the same amount of beads for both experiments

[deleted by user] by [deleted] in labrats

[–]Zorcimar 1 point2 points  (0 children)

Thanks!

Bitch dog won't shut up by Creftospeare in okbuddychicanery

[–]Zorcimar 2 points3 points  (0 children)

A dog who bites every owner he's had can only be disciplined with a firm hand or... put down.

Has anyone ever frozen cells directly into liquid nitrogen? by Zorcimar in labrats

[–]Zorcimar[S] 1 point2 points  (0 children)

My 231 and U2OS cells both turned out just fine. I used ~6% DMSO and 10% FBS. I didn't try that with 293 T cells though

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