Why are my cell lysate sample not running properly.
This is ponsue staining just after transfer. I did coomasee stained of my SDS PAGE gel and have seen similar effect. Acn anyone suggest what is happening and whats the solution?
It looks like there could be an issue with the gel polymerisation or the samples? Do you make the gels yourself? How do you collect the samples, do they all have the same salt concentration?
Gautam Agarwal < Why are my cell lysate sample not running properly. >
Can you explain which part was not running properly? I could not detect it..
Dear Holly,
Its ready made gel from Biorad. Sample was centrifuged at 15000g for 30min before collection and protein estimation. Salt concentration is same for all samples.
Dear Yuan,
You can see prominent bulg at the centre of the blot. That should have run completely.
Did you get bubbles out from the bottom area between the glass plates before running the gel?
Firstly a blot could be define as a
spot or stain,especially of ink on paper a blemish on a persons character or reputation .He had been haunted by a blot on his part or a soiling or disfiguring mark;spot a mark of reproach ,a moral flaw a usually nitrocellullose or nylon sheet that contains spots of nitrocellulose (as of DNA ,RNA or protein )or their fragments and is used to identify specific components of the spots by applying a molecular nucleic acid or a radio labelled antibody ).To spatter with a discolouring substance ,to stain with an absorbing agent ,To remove with absorbing agent ,To remove with absorbing material,a lone backgammon man exposed point ,a reputation ,disagrace ,memories blotted from one's mind.
Problems with my blot.
(1)improper assembly of transfer sandwich ,trapped air bubbles
(2)over heated buffet
(3)Dried out improperly hydrated membrane .
(4)incomplete blocking of membrane.
(5)Contaminated blocking reagent
(6)lncubation with substrate for too long .
(7)Too much antibody .
(8)Contamination in incubation tray.
(9)Excessive protein loading
(10)Too much SDS in transfer buffer.
(11)Exposure too long
(12)Blotting procedure incubation
(13)Antibody activity loss due to long term or improper storage .
(14)lncubation temperature too high .
(15)insufficient concentration of detergent in the buffers.
(16)Parts of the membrane dried out .
(17)Contamination on blot
(18)lnsufficient access of washing or incubation buffer to areas of the blot .
Aggregated secondary antibody .
(19)Gel pieces stuck to membrane .
(20) Gel pieces stuck to membrane
(21)Dirty scanned or cassette surfaces .
(22)Buffer contamination
(23)Foam pads are contamination or too thin
(24) Transfer buffer contaminated.
Hello Aristobolo,
no there was no bubbles before the run was started.
Gautam Agarwal I still think the run was normal, unless I misunderstand you. I got this pattern too-- a 'large' fat bands. Was that what you meant? See attachment, 'large' fat bands shown. It is to do with the primary antibody... see attachment and link below. (click to view the whole Fig.)
https://www.rndsystems.com/resources/technical/troubleshooting-guide-western-blot-figures
Gautam Agarwal Or, did you mean the 'bright white' thing in the picture? Originally, I thought that was a reflection from light.
- Badges
- Science method