Staining the membrane:Stain the membrane after the transfer to evaluate thetransfer efficiency. If you are using the membrane forpeptide sequencing, you may stain the membrane withCoomassie Blue stain.
If you are using the membrane for immunodetection, youcan stain the membrane with a temporary stain such asPonceau S see page 18 for Ponceau S staining. Aftervisualizing the transferred protein bands on the membrane,you can rinse the membrane with deionized water tocompletely remove the staining or incubate the membranedirectly in the blocking solution.
Ponceau S stain does notinterfere with most immunodetection methods. Optimizing Blotting ParametersIntroductionEach parameter of the blotting protocol plays a role duringthe transfer of proteins. An ideal blotting protocol balanceseach parameter to provide efficient transfer of proteins. When using the XCell II Blot module, most proteins willtransfer efficiently using the protocol on page Based on specific properties of a protein or a set of proteins,some optimization of the blotting protocol may be necessary.
Review the points described below to optimize the blottingprotocol. GelPercentageChoose the lowest percentage acrylamide appropriate for themolecular weight of your protein or proteins of interest. Gradient gels are excellent for blotting a range of proteinsizes, as the porosity of the gel matrix is well matched withthe different sizes of the proteins.
Some proteins can be equally well resolved on a Tricine gelor Tris-Glycine gel. In general, a Tricine gel will resolve thesame range of proteins as a higher percentage Tris-Glycinegel and can be a better choice for some transfers. GelThicknessThe 1. Be sure to scaleyour sample load appropriately for the sensitivity of yourantibody detection method.
Our recommended conditionfor most proteins is 25 Volts for 90 minutes. You may makeminor adjustments 5—10V accordingly, when necessary.
See Transfer Conditions. Alcohol inTransferBufferDecreasing or eliminating alcohol may improve the transferof some proteins, especially large proteins. This is balanced by the residual SDS in the gel fromthe running buffer. Keep this in mind when adjusting themethanol content in transfer buffer.
Optimizing Blotting Parameters, ContinuedTransferTimeIncreasing the transfer time to two hours will improvetransfer of most proteins, but may cause the smaller proteinsto pass through the membrane.
Transfer time usually haslittle influence on the detection of proteins that remainbound to the membrane. Note that transfer times longerthan 2 hours at the recommended power settings do notgreatly improve transfer of proteins that have failed totransfer completely in 2 hours.
This may be due to theexhaustion of the buffer or partial fixation of the protein inthe gel as a result of the removal of SDS or a conformationalchange in the proteins during the transfer interval.
In the blotting protocol on page 12, the gel is not incubatedin the transfer buffer, leaving residual SDS in the gel. Keep this in mind when adjusting the SDS content in thetransfer buffer. Charge ofProteinFor more basic proteins, use a carbonate buffer.
The high pHof the buffer confers a higher negative charge on the morebasic proteins and cause them to migrate faster. Carbonatebuffers improve binding detection for some systems.
Due tothe high ionic strength of the carbonate buffer, excessiveheat may be generated during blotting. HintWe recommend using two membranes in tandem duringinitial blotting to closely monitor the protein transfer andthen perform the same visualization technique on bothmembranes. Monitor whether the primary membranelocated next to the gel retains majority of the sample. If thesample is detected on the membrane placed closer to theanode further away from the gel , reduce the rate of transferby lowering the field strength, allowing more time forprotein capture on the primary membrane.
Adjust theblotting protocol accordingly using the guidelines includedin this section. TroubleshootingIntroductionProblemReview the information provided below to troubleshootyour experiments. Significantamount ofprotein ispassing throughthe membraneindicated by thepresence ofproteins on thesecondmembraneLonger transfer time,inappropriate SDS ormethanol content, orsample overloaded Re-evaluate the percentageof the gel used.
Shorten the transfer time by15 minute increments. Remove any SDS which mayhave been added to thetransfer buffer. If using nitrocellulosemembrane, switch to PVDFwhich has a higher bindingcapacity. Add additional methanol toincrease the binding capacityof the membrane. Decrease the sample load. Switch to a more appropriatelower percentage gel. Increase the blotting time by15 minute increments. Add 0. Decrease the amount ofmethanol in the transferbuffer. Significantamount ofprotein remainsin the gelindicated bystaining of thegel after transferShorter transfer time,inappropriate gel type,SDS or methanol contentHigher molecular weightproteins usually do nottransfer completely ascompared to mid to lowmolecular weightproteinsContinued on next page Do not adjust the pH with acidor base as this will increase theconductivity of the buffer andresult in higher current duringtransfer.
Current is muchhigher than theexpected startcurrentConcentrated buffer usedDilute the buffer as described onpage 8. Used Tris HCl instead ofTris BaseCheck the reagents used to makethe buffer and remake the bufferwith correct reagents. Current is muchlower than theexpected startcurrentVery dilute buffer usedresulting in increasedresistance and lowcurrentRemake the transfer buffercorrectly.
The circuit is broken broken electrode Check the blot module to ensurethat the electrodes are intact. Leak in the blot moduleindicated by a decrease inthe buffer volume in themoduleBe sure to assemble the blotmodule correctly to prevent anyleaking.
High ionic strength of thetransfer bufferPrepare the buffer as describedon page 8. Power supply isoperating at a currentclose to the current limitof the power supplyUse a power supply with higherlimits. Power supplyshuts off usingrecommendedblottingconditionsContinued on next page Saturate the blotting pads withtransfer buffer to remove airbubbles. Southern, or northern transfer of two mini-gels using only mL of transfer buffer. An efficient transfer is obtained, as the resistance is constant across the blotting electrodes producing uniform field strength.
Continued on next page. Export To Word. Last View : 7d ago. Upload by : Alexia Money. Report this link. Related Books. Remove any trapped air bubbles Place two soaked blotting pads into the cathode - core of the blot module. The cathode core is the deeper of the two cores and the corresponding electrode plate is a darker shade of gray. Carefully pick up the gel membrane assembly and place on blotting pad in the same sequence, such that the gel is closest to the cathode core follow manual Add enough pre-soaked blotting pads to rise to 0.
Blot Module to fit horizontally across the bottom of the unit. There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. Properly placed, the inverted gold post on the right hand side of the blot module will fit into the hole next to the upright gold post on the right side of the lower buffer chamber Place the Gel Tension Wedge so that its vertical face is against the blot module.
Do not fill all the way to the top as this will only generate extra conductivity and heat Fill the Outer Buffer Chamber with deionized water by pouring approximately ml in the gap between the front of the blot module and the front of the lower buffer chamber. The water level should reach approximately 2 cm from the top of the lower buffer chamber. This serves to dissipate heat produced during the run Place the lid on top of the unit With the power turned off, plug the red and black leads into the power supply.
Mix 6 mL of Detection Reagent 1 and 2 for each transfer membrane Pour over transfer membrane and leave on for one minute. Dry off with Kimwipe Bring to the imager. Stock Quick Quote. Special financing available Select PayPal Credit at checkout to have the option to pay over time.
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