Antibody common problem of WB

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Antibody common problem of WB

Problem	Reason	Solution
The strip shape is not good	Uneven gelation, poor polymerization	Mix the solution thoroughly before filling
	Distorted loading teeth make loading holes different in size	Use the needle to straighten the teeth before loading the sample, be careful not to insert the needle into the glue
	Some samples have higher salt concentration	Desalt or adjust the sample salt concentration to the same
	Inconsistent sample volume per well	Adjust the sample amount to make it basically the same
	Buffer is outdated, composition has changed	Reconfiguration
	There are bubbles under the gel	Remove bubbles before electrophoresis
	The temperature is too high during electrophoresis	Reduce current or voltage
Weak signal of target protein	The sample load is insufficient or the target protein concentration is too low	Increase sample load or concentrate sample
	Low transfer efficiency	Single column below
	Low antibody concentration	Increase antibody concentration or extend incubation time
	Insufficient concentration of developer substrate	Increase the amount of developer
	Chromogenic agent failure	Change the developer (the tips for sucking A and B liquid can not be mixed)
	Insufficient color development or exposure time	Extend color development or exposure time
Low transfer efficiency	The pH value of the transfer buffer is close to the isoelectric point of the target protein	Increase the pH value of transfer buffer
	There are bubbles between the gel and the membrane	Drain the bubbles before transferring the film
	Large area contact between the gel and the filter paper on both sides of the transfer membrane	The size of filter paper, transfer membrane and gel should be basically the same, avoid direct contact with filter paper
	Improper choice of transfer film type	Use reliable PVDF membrane or nitrocellulose membrane
	Voltage or current is too small	20mA constant current during wet running, constant voltage around 25V during semi-dry running
	The transfer time is too long or too short, the protein is still in the gel or penetrates the transfer membrane	Electrophoresis first, select the appropriate transfer time according to the size of the protein and the transfer device
	The ambient temperature is too high during wet transfer	Use pre-cooled transfer buffer or set the device to 4 degrees
No banding after color development or exposure	Glue and film are in the opposite direction	When transferring the membrane, the electrodes corresponding to the PDVF film and the gel should be placed in sequence, that is, the gel corresponds to the negative electrode and the PDVF film corresponds to the positive electrode.
	The selected primary antibody, secondary antibody and color rendering method are inappropriate	Choose the appropriate primary antibody, secondary antibody and color development method
	The target protein content is below the lower limit of detection	Increase sample load or concentrate sample
	Antibody titer is too low	Increase antibody concentration
	Insufficient antibody incubation time	Extend the incubation time, incubate at 37°C for more than 1 hour
	Antibody overwashing	Reduce washing time and frequency
	The interval between adding HRP substrate reaction and exposure detection is too long	Response 3 to 5 minutes, timely detection
Background is too high	Insufficient amount of closure	Increase the concentration of the blocking material and ensure that the blocking solution is completely immersed in the transfer membrane during incubation
	Improper use of closures	Can not be blocked with skimmed milk powder when detecting biotin-labeled protein
	Not enough closed time	Seal at room temperature 37°C for more than 1 hour, lock at 4°C overnight
	Antibody non-specific binding	Reduce antibody concentration and reduce incubation time
	The antibody concentration is too high or the washing is not enough	Decrease antibody concentration, increase washing times and time
	Too much chemical chromogenic substrate	Add appropriate amount of chromogenic substrate according to the instructions
Miscellaneous	More protein	Processing target protein
	Antibody is not specific	Use specific antibodies
	Antibody incubation time is too long	Reduce antibody incubation time
	The secondary antibody cross-reacts with the antigen	Choose the right closure
	The color development and exposure time of the substrate is long	Shorten the time of color development and exposure
Large molecular weight WB	Membrane pore size is too small	Replace membrane with larger pore size
	Low transfer voltage and current	Increase voltage/current
	Short transfer time	Extend transfer time
	Glue concentration is too high	Use low concentration glue
	Improper transfer buffer formulation	Adjust the concentration of methanol and SDS in the transfer buffer

Antibody common problem of WB

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