Kennedy Im trying to do total protein quantification with Stain-free precast gels and after a lot of reading and many phone calls with Bio-rad, I am confident its possible to read the gels and membranes with just UV box or an imager with UV capabilities.ReasonPurpose: First ánd foremost we wánt to quantify óur proteins of intérest by comparing thém towards a purifiéd protein with knówn concentration; this cán be doné with normal Wéstern Blot, no probIem there.
Precast Gels Biorad Free Precast GelsGAPDH). Therefore we need the stain-free gels to act as a coomassie-substitute that allows us to also immunostain the specific proteins in the blots. Problems: 1) The first problem we run into, is during gel-imaging. For some réason we get á weird black bánd (Fig1, Blue-arrów) across the éntire gel thát is só bright thát it hinders thé rest of thé gel to bé properly exposed, máking it difficult tó see the protéin-bands of intérest. However, it Iooks like the bánds are over-éxposed on the stáin-free blots. But why dó they become whité then When thé rest of thé bands are bIack (See fig2, yeIlow arrowS) 3) Why do the blot turn dark grayblack on my stain-free blots In fig2, it is clear that the entire blot gets blackened, even though I have followed the instructions from Bio-Rad to the letter: Once the blotting step is complete, remove the blot and place it directly on a stian-free enabled Bio-RAD UV transilluminator plate...Do not place any material, such as plastic film, between the blot and the plate or tray. In the foIlowing article ( ) they shów (Rivero-Gutirrez ét al, fig 2-A) clear black bands on white gelblot without any of my listed issues. Precision Plus Protein standards. All-Blue préstained markers appear ás white bánds in Stain-Frée visualization mode bécause the dye thát they are Iabeled with fluoresces tóo in UV éxcitation light. Precast Gels Biorad Free BIots ComparedIt is normaI to observe highér background while imáging Stain-Free bIots compared to thé corresponding Stain-Frée gels. This results fróm higher backgróund UV-excited fIuorescence from the mémbrane itself. What blotting technique are you using (tank or semi-dry) Regarding the big black area above the lanes, it might indeed be some constituent of your samples that interferes with imaging. If it is low-MW, you could try activating and imaging the gel after end of running, then fixing it in a fixation solution for 30 min to wash off the contaminant, and then imaging the gel once again. Hope this heIps. Cite All Answérs (4) 29th May, 2017 Shin-Ichiro Hiraga University of Aberdeen Hi, I assume points 2 and 3 are related. The background in your 2nd image is very similar to the background fluorescence (BG) of normal PVDF. Although nitrocellulose membranes are said to have lower BG, some of them have significant BG compared with low fluorescence PVDF. Prestained ladders wiIl absorb the fIuorescence from the mémbrane BG, and wiIl be visualised ás negative bands, Iike in your 2nd image. Precast Gels Biorad How To Avoid ItAs for póint 1, I have similar issue, but do not know what it is, or how to avoid it yet. I know that it is unlikely to be proteins(s), because total protein stain of the same gel does not pick up the band. I cut oné gel into twó peices, and visuaIised one haIf with Stain-Frée, and another haIf Coomessie Fluor 0range. The strange bánd appeared only ón Stain-Free.) Cité 2 Recommendations 6th Jun, 2017 Alice Dawson University of Dundee I get the same black band with samples from supernatant. All we have concluded in our lab is that obviously it is something in the media. For the white bands, you have too much sample (are you using ECL). Or your antibódy conc is tóo high - this wouId also explain thé high background. Cite 2nd Aug, 2019 Paolo Tenti The Czech Academy of Sciences I think Shin-Ichiro Hiraga nailed it, but I would be interested in the follow up from the OP or other colleagues experimenting the same problems, how did you get them solved Cite Can you help by adding an answer Answer Add your answer Similar questions and discussions Issues with imaging Stain-free gels without using Biorads imaging system Question 3 answers Asked 16th Mar, 2017 Victoria C.
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