WB help

May 15, 2020 0 By Mashid

The next information serves as a guidelines for the attainable causes and options with respect to a number of the mostly encountered issues from the Western blot assays.

Excessive Background

Potential Trigger Resolution

1 Too excessive antibody focus

Optimize and reduce antibody focus

2 Combination secondary antibody formation

Filter the secondary antibody by means of 0.2μm filter

Use a brand new secondary antibody

three Too high antibody incubation temperature

Incubate the antibody at 4°C

Four Non-specific secondary antibody binding or cross-reactivity with blocking agent

Run secondary antibody management (with out the first)

Lower secondary antibody concentration

5 Cross-reactivity of main or secondary antibody with blocking agent

Add Tween-20 to the incubation and washing buffer

6 Incompatible blocking agent

Evaluate completely different blocking buffers

7 Incomplete blocking

Optimize selection of blocking buffer

Improve protein focus in blocking agent

Optimize blocking time and/or temperature; Block for two hours at regular temperature or in a single day at 4°C

Add 0.05% Tween 20 detergent into blocking agent

Add 0.05% Tween 20 detergent into antibody diluents resolution

eight Inadequate blocking

Lengthen blocking time or use a appropriate blocking agent (e.g. skim milk, BSA, serum, and so on.)

9 Cross-reactivity of antibody with different proteins

Use completely different blocking agent (Don’t use skim milk with biotin system

Scale back secondary antibody focus

Check cross-reactivity between secondary antibody and membrane

10 Inadequate washing

Improve variety of washes and buffer quantity

Add 0.05% Tween 20 detergent into washing buffer

11 Too lengthy publicity time

Scale back publicity time

12 Membrane drawback

Use clear tweezers; Function with gloves

Use new membranes

Make sure the liquid is sufficient to maintain the membrane moist

Use decolorization desk in incubation

Keep away from membranes overlapping

Deal with rigorously and keep away from damaging membrane

13 Inadequate membrane wash

Improve the variety of wash

14 Incompatible membrane

Nitrocellulose membrane’s background is decrease than that of PVDF membrane

15 Dry membrane

Be certain that membrane is roofed with a adequate quantity of liquid and forestall it from drying

16 Contaminated buffer

Use new buffer or filter buffer earlier than use

17 Contaminated tools

Guarantee all tools and instruments are clear and no gel is left on membrane

Weak/No Sign

Potential Trigger Resolution


1 Improper protein switch to membrane
Stain gel after switch is full to find out switch is environment friendly
Use Ponceau S to stain membrane to find out switch is environment friendly
Guarantee adequate contact between gel and membrane throughout switch
Be certain that switch sandwich is assembled appropriately
Moist membrane in accordance the instruction
Keep away from overheating throughout electro-transfer
Use optimistic management or molecular weight markers
Optimize switch time and present
Use Boster’s Membrane-Transferring Buffer (AR1149)
Keep away from pattern (antigenic determinant) destroy when dealing with
2 Inadequate protein and membrane binding
Including 20% methanol to switch buffer
Use small-bore membrane
three Inadequate antibody
Improve antibody focus
Four Inadequate antigen
Load extra protein
5 Antigen masking by blocking buffer
Evaluate completely different blocking buffers
Optimize protein focus of blocking agent
Scale back blocking time
6 Presence of sodium azide in buffers
Remove sodium azide from buffers
7 Too brief publicity time
Lengthen movie publicity time
eight Too brief substrate incubation time
Lengthen substrate incubation time to 5 minutes
9 Digestion of protein on membrane
Optimize quantity of blocking agent
10 Degradation of protein throughout storage
Re-prepare protein pattern
11 Incompatible main and secondary antibodies
Be certain that main antibody, secondary antibody, substrate, enzyme system and samples are appropriate
Use loading management to check effectiveness of second detecting system
12 Low focus of main antibody and/or secondary antibody
Improve antibody focus
Improve incubation time
13 Cross-reactivity between blocking agent and antibodies (main or secondary)
Use delicate detergent equivalent to Tween20
Change blocking agent (generally used are milk, BSA, serum or gelatin)
14 Incapability of main antibody to acknowledge the protein in examined pattern
Examine instruction
Use optimistic management
15 Low or none content material of goal protein (ineffective antigen)
Use optimistic management
Improve loading quantity to 20-30 µg protein per effectively
Use protease inhibitor or fractional extract goal protein
16 Inadequate switch and extreme wash
Examine the switch with Ponceau S
Soak PVDF-membrane in methanol
Keep away from extreme wash
17 Over-blocking
Use 0.05% skim milk or no milk diluents buffer
Change blocking agent
Scale back blocking time
18 Lack of main antibody effectiveness
Put together contemporary antibody and retailer correctly when not in use
Keep away from repeated freezing and thawing
19 Inhibition of secondary antibody by sodium azide
Keep away from utilizing sodium azide along with HRP- conjugated antibodies
20 Lack of effectiveness in enzyme conjugate and substrate
Combine enzyme conjugate and substrate (no coloration improvement when enzyme is inactive)
Use activated enzyme conjugate and contemporary substrate
21 Improper moist switch for membrane
Soak PVDF membrane in 100% methanol
22 Inadequate molecular weight of goal protein (< 10 kDa)
Use small-bore membrane
Scale back switch time
23 Equality or nearness in values between goal protein’s isoelectric level and switch buffer’s pH worth
Strive different buffers equivalent to CAPS buffer (pH 10.5)
Strive low pH worth buffers equivalent to acetic acid buffer
24 Too excessive methanol focus
Lower methanol focus or use isopropyl alcohol