It is important. Do you have to use methanol? So why all the fussiness? It seems to come down to purity and time. Purity You always want to use high quality alcohol for membrane activation. Time If you are going to use an alcohol other than methanol to activate the membrane, then add a few extra minutes into your protocol.
Leave a Reply Cancel reply Your email address will not be published. Contents Visualization of proteins in gels Transfer Visualization of proteins in membrane with Ponceau Red Blocking the membrane Incubation with primary antibody Incubation with secondary antibody Development methods. Visualization of proteins in gels Protein visualization at this stage is useful to determine if proteins have migrated uniformly and evenly.
Coomassie stain As soon as the power is turned off the separated protein bands will begin to diffuse they are freely soluble in aqueous solution. Copper stain Briefly rinse freshly-electrophoresed gels in distilled water 30 sec maximum and then transfer to a solution of 0. Transfer of large and small proteins The balance of SDS and methanol in the transfer buffer, protein size, and gel percentage can affect transfer efficiency.
These will be very fragile, so handle carefully. Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0. Lowering the methanol percentage in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily.
Methanol is only necessary if using nitrocellulose. If your protein of interest is small, omit SDS from transfer buffer. The following reference discusses a gel and buffer system that allows transfer of proteins as large as kD: Bolt MW and Mahoney PA More transfer tips: Avoid touching the membrane with your fingers; use tweezers instead. Oils and proteins on fingers will block efficient transfer and create dirty blots. After sandwiching the gel and membrane between paper, air bubbles between the gel and membrane can be removed by rolling them out with a roller, pipette or 15 mL tube, or by assembling the sandwich in a dish of transfer buffer to prevent formation of bubbles in the first place.
Make sure the paper and membrane are cut to the same size as the gel. Large overhangs may prevent a current from passing through the membrane in semi-dry transfers. Chicken antibodies tend to bind PVDF and other nylon-based membranes, leading to high background.
Switching to a nitrocellulose membrane should help reduce background staining. TBS 10x For 1 L; Rinse for 5 s in TBST after the incubation.
Incubation time The time can vary between a few hours to overnight rarely more than 18 h , and is dependent on the binding affinity of the antibody for the protein and the abundance of protein. Incubation with secondary antibody Wash the membrane several times in TBST while agitating, 5 min or more per wash, to remove residual primary antibody. Incubation time and temperature 1—2 h at room temperature with agitation. Which conjugate? X-ray films Automated x-ray film developers are widely used and easy to use.
Digital images The new generation of film developers are units with a camera inside an enclosure, removing the need for a darkroom. Return to western blot protocols. Contaminated dishes, boxes, or trays Clean dishes, boxes, or trays with methanol before using them for incubation. Dirty scanning surface or silicone mat Clean scanning surface and mat carefully before each use. Marks on membrane from incompatible marker or pen Use pencil to mark membranes. Dirty transfer pads or transfer box Transfer pads in wet tank systems and transfer boxes accumulate residue after frequent use that can cause speckles on Western blot membranes.
Dirty imaging system Always clean your imaging system before you image. Not enough antibody used Primary antibody may have low affinity for your target. Degraded antibody Primary and secondary antibodies can lose reactivity from improper or extended storage. Signal washed away by too much detergent Decrease Tween 20 in diluted antibodies. Recommended Tween 20 concentration is 0.
For nitrocellulose membranes, do not add SDS to any steps. Unsuitable blocking buffer for experiment Your primary antibody may work much better with a different blocking buffer. Inappropriate antigen amount loaded Loading too little or loading too much protein sample will decrease antibody sensitivity. Protein did not transfer well Check transfer buffer choice and blotting procedure.
Protein lost from membrane during detection After transfer is complete, dry your membrane before you block. For PVDF membranes, re-activate membranes with methanol and rinse with water before blocking.
Proteins not retained on membrane during transfer SDS in transfer buffer may interfere with binding of transferred proteins, especially for low molecular weight proteins. Over-blocking the membrane Extended blocking times can mask antigen and decrease signal intensities.
Unsuitable blocking buffer for experiment The blocker you use may affect background bands. Antibody cross-reactivity in a two-color Western blot In two-color Western blots, antibody cross-reactivity is always a possibility. To avoid cross-reactivity: Detect the two secondary antibodies on two separate blots first. See what each channel looks like individually before you detect the two secondary antibodies together on the same blot, to help you know what bands to expect and where to expect them.
Use secondary antibodies from the same host species to avoid potential cross-reactivity. Avoid using mouse and rat primary antibodies together if possible. Because the species are so closely related, anti-mouse will react with rat IgG to some extent, and anti-rat with mouse IgG. Sheep and goat antibodies will also cross-react. Use less secondary antibody to minimize cross-reactivity — a good starting dilution is , Dilute between , — , for optimal performance.
Reduce antibody incubation time to 1 hour at room temperature. Dilute antibodies in the same blocking solution that you used to block the blot. Protein estimation can be performed using as little as 0. Why do PVDF membranes require a methanol soak? Nitrocellulose membranes are hydrophilic so can be fully hydrated by aqueous buffers.
0コメント