option 1 and 2 for estimating mw in R. option 1 is manual measure, option 2 is using locator()

Author: 3mmaRand

r4babs2/week-2/data-image/26C-10-Coomassie-Blue.jpg

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+kda
+250
+130
+100
+70
+55
+35
+25
+15
+10

r4babs2/week-2/data-image/sds-page-gel.png

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+kda,dist_to_band
+250,138
+130,213
+100,270
+70,309
+55,356
+35,435
+25,478
+15,563
+10,623

r4babs2/week-2/data-image/standard-mw.txt

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+library(tidyverse)
+library(imager)
+
+# import gel
+gel <- load.image("data-image/26C-10-Coomassie-Blue.jpg")
+
+# import molecular weights
+mw <- read_table("data-image/standard-mw.txt")
+
+plot(gel)
+
+# crop
+gel_cropped <- crop.borders(gel, nx = 300, ny = 150)
+
+plot(gel_cropped)
+
+gel_top <- 180
+gel_bottom <- 990
+
+# add a line to plot 
+abline(h = gel_top, col = "red")
+abline(h = gel_bottom, col = "red")
+
+# vertical guides just to help with locator
+abline(v = 220, col = "red")
+abline(v = 510, col = "red")
+
+gel_length <- gel_bottom - gel_top
+
+# Number of bands in your marker lane
+# click from the top or gel to the bottom 
+# i.e., high MW to low
+marker_positions <- locator(n = 9)  
+
+pos_patB <- locator(n = 1)
+dist_to_patB <- pos_patB$y - 180
+patB_Rf = (gel_length - dist_to_patB) / gel_length
+
+
+# add marker positions to the mw data frame
+mw <- mw |> 
+  mutate(x = marker_positions$x,
+         y = marker_positions$y,
+         dist_to_band = y - 180,
+         Rf = (gel_length - dist_to_band) / gel_length,
+         log_kda = log(kda)) 
+
+
+ggplot(data = mw, aes(x = Rf, y = log_kda)) +
+  geom_point(size = 4) +
+  geom_smooth(method = "lm", 
+              se = FALSE,
+              colour = "gray30") +
+  theme_classic()
+
+# fit a linear model
+mod <- lm(log_kda ~ Rf, data = mw)
+
+patB_log_kda <- predict(mod, newdata = data.frame(Rf = patB_Rf)) 
+
+
+ggplot(data = mw, aes(x = Rf, y = log_kda)) +
+  geom_point(size = 3) +
+  geom_smooth(method = "lm", 
+              se = FALSE,
+              colour = "gray30") +
+  annotate("segment", x = patB_Rf, xend = patB_Rf, 
+           y = 0, yend = patB_log_kda,
+           linetype = "dashed", 
+           colour = "red") +
+  annotate("segment", x = 0, xend = patB_Rf, 
+           y = patB_log_kda, yend = patB_log_kda,
+           linetype = "dashed", 
+           colour = "red") +
+  scale_x_continuous(expand = c(0, 0),
+                     limits = c(0, 1)) +
+  scale_y_continuous(expand = c(0, 0),
+                     limits = c(0, 6)) +
+  theme_classic()
+
+
+
+patB_kda <- exp(patB_log_kda) 
+
+
+# check results are the same.
+# option one
+# > mw_positions
+# # A tibble: 9 × 2
+# kda dist_to_band
+# <dbl>        <dbl>
+#   1   250          138
+# 2   130          213
+# 3   100          270
+# 4    70          309
+# 5    55          356
+# 6    35          435
+# 7    25          478
+# 8    15          563
+# 9    10          623
+
+# gel_length <- 810
+# pos_patB <- 394
+
+# mw_positions
+# # A tibble: 9 × 4
+# kda     dist_to_band    Rf log_kda
+#   <dbl>        <dbl> <dbl>   <dbl>
+# 1   250          138 0.830    5.52
+# 2   130          213 0.737    4.87
+# 3   100          270 0.667    4.61
+# 4    70          309 0.619    4.25
+# 5    55          356 0.560    4.01
+# 6    35          435 0.463    3.56
+# 7    25          478 0.410    3.22
+# 8    15          563 0.305    2.71
+# 9    10          623 0.231    2.30
+# Coefficients:
+  # (Intercept)           Rf  
+# 1.091        5.231  
+
+
+# patB_Rf
+# [1] 0.5135802
+
+# > patB_kda
+# 1 
+# 43.69469 
+
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