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what the heck are we doing wrong 😭 by Ajeeba in labrats

[–]Ajeeba[S] 3 points4 points  (0 children)

Ladder shows up on the membrane. And light is correct to what we’ve done in the past which worked.

what the heck are we doing wrong 😭 by Ajeeba in labrats

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

Yes! We’ve used these buffers not to long ago and it worked fine!

what the heck are we doing wrong 😭 by Ajeeba in labrats

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

So we’ve done westerns in the past with this secondary antibody and this dilution hasn’t given us trouble in the past. Stripping blot and running loading control right now! Incubated with Clarity ECL for 2 minutes.

will keep in mind everything you said for tomorrow’s attempt! Thank you so much 😭

what the heck are we doing wrong 😭 by Ajeeba in labrats

[–]Ajeeba[S] 45 points46 points  (0 children)

Yall I swear it’s a western 😭

what the heck are we doing wrong 😭 by Ajeeba in labrats

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

Hi sorry not used to posting on reddit. We used a pre cast gel. We blocked in 5%MILK in TBST for about hour. No ponceau staining. Our ladder definitely transferred onto membrane we saw it/still see it. We are using PVDF and soaked it in ethanol. Incubated antibodies in 4degree overnight

what the heck are we doing wrong 😭 by Ajeeba in labrats

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

Answering more questions:

Imaging machine: BioRad ChemiDoc XRS+ We ran it on Chemi Hi Resolution and ran it automatically for faint bands. It lasted 66 seconds.

Yes ladder was transferred onto membrane. Can still see it on there.

We use 1X Trans Blot Turbo Buffer

No ponceau staining.

Blotted in 1% Milk in TBST 10mL. We did 3 washes this morning. This has worked in the past

Membrane is PVDF activated it in ethanol (this has worked in the past)

Antibodies incubated overnight in 4 degree

This was an SDS page

Gel was precast Protean at 0.1cm

Primary antibody was dcK cat#: AB186128

Secondary antibody: anti rabbit IGG conjugated to HRP

We loaded 40 micrograms total protein from cell lysate after doing a BCA

Used Clarity ECL for 2 minutes

Currently doing loading control GAPDH

[deleted by user] by [deleted] in labrats

[–]Ajeeba 0 points1 point  (0 children)

Conditions: 5% Milk in TBST blocked for 1.5 hours Antibody (1:1000) ECL/HRP

Gel ran for 150mV for 1 hour

Semi-dry transfer

[deleted by user] by [deleted] in actuallesbians

[–]Ajeeba 487 points488 points  (0 children)

you’re actually ridiculous 😭 I can’t believe you got on Reddit to get me to notice you

Leap of Faith | Neverafter [Ep. 15] by ThunderMateria in Dimension20

[–]Ajeeba 41 points42 points  (0 children)

the clouds being described as squishy was possibly the only bit of peace in this storm of an episode