#Prestained protein ladder western blot serial#

The protein was transferred to a PVDF membrane using the Pierce Power Blotter ( Cat. The resulting reaction mixture was prepared for reducing SDS-PAGE, serially diluted, and electrophoresed on a Novex WedgeWell 4–20% Tris-glycine gel ( Cat. 88890) and appropriate expression-ready clones. HA-tagged RB-1 was expressed in HeLa cell extract using the 1-Step Human High-Yield Mini IVT Kit ( Cat. Up to four different proteins can be imaged simultaneously on the same blot. Four-channel imaging of a multiplexed fluorescent western blot. Clean the imager surface with ethanol to remove dust, lint, and residues before placing a blot for imagingįigure 2.Clean transfer devices and dusty consumables (e.g., pads) if using a wet transfer method as they can introduce speckles.Use clean incubation trays or dishes-rinsing with methanol followed by water will help dissolve residual dried dyes from previous uses.Use clean forceps to handle the membrane, and avoid directly touching the membrane particulates and contaminants from unclean tools may fluoresce.The membrane type can affect the background for example, PVDF membranes can autofluoresce and cause high background, so use low-fluorescence PVDF membranes.Ensure consistent agitation during every incubation step.Ensure good coverage of the whole blot during all incubation steps.Optimize the secondary antibody dilution depending on the dye being used, following the vendor's recommended dilution and adapting accordinglyīlotchy or uneven background due to the membrane drying out.High background from an excess of secondary antibody Increase the number or duration of wash steps.Prepare the secondary antibody diluent with 0.05% Tween 20 detergent.Use a wash buffer with 0.1–0.2% Tween 20 detergent.Load less of the molecular weight marker onto the gel.

Determine the best blocking buffer for your application-primary antibodies will react differently in different blocking buffers blocking buffers like normal animal sera or milk may result in cross-reactivityĪrtifacts from overloading the protein marker or ladder.Handle the membrane carefully using clean dishes or trays and clean forceps.High background due to membrane contamination Use the autoexposure feature on the instrument to determine the optimal exposure time for each channelīackground issues (high, uneven, or speckled).Ensure that your fluorescent dyes can be distinctively detected on your imager.Usa a tool like Fluorescence SpectraViewer to visualize fluorphore spectra, and avoid spectrally close conjugates, especially when the signal is very strong.Reduce the amount of the secondary antibody used to remain within the optimal performance rangeįluorescent bleed-through from another channel when multiplexing (appearance of an unexpected band).Use highly cross-adsorbed secondary antibodies.Choose primary antibodies raised in distantly related species.Sample degradation due to overheating or protease activity results in target breakdown and low target recognition by the antibodyĪntibody cross-reactivity in multiplex detection.Evaluate additional primary antibodies find tips at /antibodyvalidation.Poor antibody specificity for the target of interest Reoptimization may be required when probing for a new protein.Check transfer conditions to confirm protein transfer.Poor transfer of protein or loss of the protein after transfer

#Prestained protein ladder western blot serial#

Perform serial dilutions of the lysate or sample to determine the optimal amount of protein to load.

Too little lysate leads to insufficient availability of the target of interest.

Insufficient quantity of sample loaded on the gel

Dilute your primary antibody in wash buffer.

Some blocking solutions can mask the blot and reduce the availability of the antigen to the antibody, especially if the blocking step is >1 hour.

Too much detergent or the nature of the detergent can result in washing away the signal-decrease or eliminate detergent.

Ensure the correct excitation and emission ranges are selected for the intended fluorophore.

Use the Smart Exposure feature on the iBright FL1500 system, or a comparable autoexposure feature on another instrument.

Avoid multiple uses of pre-diluted antibodies.

Check the expiration date of the antibody.

Ensure the antibody was stored appropriately.

Extend the incubation time to overnight at 4☌, or 3–6 hours at room temperature.

Ensure primary antibody has a good titer and is specific for the antigen to be detected.

Increase primary antibody concentration.

Spectra Multicolor Low Range Protein Ladder Spectra Multicolor High Range Protein Ladder NuPAGE MOPS SDS Buffer (15–260 kDa) Bolt MES SDS Buffer (3.5–160 kDa) add Bolt Antioxidant for reduced samples Low-abundance/ posttranslationally modifiedīolt MOPS SDS Buffer (15–260 kDa) Bolt MES SDS Buffer (3.5–160 kDa) add Bolt Antioxidant for reduced samples