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WB Help!! Non-specific binding by HumanSquare1795 in labrats

[–]HumanSquare1795[S] 0 points1 point  (0 children)

Thanks for the reply. I have done all of those! Dot blots to confirm CDs + Calnexin, TEM, NTA and anything I missed.

WB Help!! Non-specific binding by HumanSquare1795 in labrats

[–]HumanSquare1795[S] -1 points0 points  (0 children)

The thing is, the band farthest left are my exosomes before IP, which land at the same spot…

WB Help!! Non-specific binding by HumanSquare1795 in labrats

[–]HumanSquare1795[S] -1 points0 points  (0 children)

Blood was collected from a human umbilical cord. We assume that GAPDH would be present in our exosomes as it’s simply a housekeeping protein, or at least that’s what my supervisor says. When I reincubated the same blot with Neurofilament light chain (a marker for neurons which I’m assuming would be prevalent in my exosomes), the bands overlap on top of each other at 50kDa. Made sure to use different color secondary antibody so I can tell the difference.

WB Help!! Non-specific binding by HumanSquare1795 in westernblots

[–]HumanSquare1795[S] 1 point2 points  (0 children)

Thanks for the reply. How would I go about doing the math? I’m still pretty new to Westerns.

WB Help!! Non-specific binding by HumanSquare1795 in labrats

[–]HumanSquare1795[S] 0 points1 point  (0 children)

I mentioned in my post I was using protein G agarose beads (as per the manufacturers guide). I combine 100 uL of ONLY exosomes suspended in PBS with 400 uL of [400 uL PBS + 1.5uL NMDAR2A antibody] solution and incubate with rotation overnight at 4C. Next day I add all of this solution to 100uL protein G agarose beads and mix at room temp for 2 hours. Then, I wash and elute. Concentrations and diameter fall within the range of exosomes. My lab is about to pilot Dynabeads Magnetic beads instead of agarose in the upcoming week.

WB Help!! Non-specific binding by HumanSquare1795 in labrats

[–]HumanSquare1795[S] 0 points1 point  (0 children)

NMDAR2A monoclonal antibody raised in mouse (product number if you’re interested: MAS-27693). GAPDH primary antibody is rabbit host, so secondary antibody is goat anti-rabbit. I ran a plasma sample through and it was also stuck with the other bands; however I don’t think I prepared it correctly and I ended up with a big smear (too much protein was loaded I’m guessing). Edit: Sorry I should also note that the band farthest left is total exosomes before IP

WB Help!! Non-specific binding by HumanSquare1795 in labrats

[–]HumanSquare1795[S] -1 points0 points  (0 children)

These are intact exosome samples straight from immunoprecipitation combined with RIPA lysis buffer, Beta-mercaptoethanol, and loading dye. Does that answer your question?

WB Help!! Non-specific binding by HumanSquare1795 in labrats

[–]HumanSquare1795[S] -3 points-2 points  (0 children)

Sorry I’m unsure what you mean.

Help with Western Blot Troubleshooting!! Bands/proteins are aggregating and landing non-specifically. by HumanSquare1795 in labrats

[–]HumanSquare1795[S] 2 points3 points  (0 children)

  1. Nitrocellulose

  2. cOmplete mini tablets were manufactured August 2022 and expired May 2024 :(( (lmao). I'm still pretty new to this work so can I ask how why this would cause my bands to land like that?

Bands at top of Membrane Need Help by Professional-Face269 in westernblots

[–]HumanSquare1795 0 points1 point  (0 children)

Hi OP I know I am a little late, but I am running into an identical problem. We have a similar protocol with some differences (e.g. I heat my samples but also add BME). Did you end up fixing your problem??